Lipid nanoparticle (LNP) formulations

Abstract

The present disclosure describes lipid nanoparticle (LNP) compositions and uses thereof, e.g. for treating ocular-related disorders or diseases.

Description

[0001] LIPID NANOPARTICLE (LNP) FORMULATIONS

[0002] FIELD

[0003] The present disclosure relates to lipid nanoparticles and uses thereof, e.g. for treating ocular-related disorders or diseases.

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS

[0005] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 498,437, filed on April 26, 2023 and U.S. Provisional Application No. 63 / 561,544, filed on March 5, 2024, the entire content of which are hereby incorporated herein by reference in its entirety.

[0006] SEQUENCE LISTING

[0007] The instant application contains a Sequence Listing that has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. The Sequence Listing for this application is labeled “SAL-028_SequenceListing”, which was created on April 14, 2024, and is 46,674 bytes in size.

[0008] BACKGROUND

[0009] Lipid nanoparticles (LNPs) are a type of lipid vesicles that possess a lipid core. These vesicles are used in, e.g., nucleic acid delivery.

[0010] Messenger RNA (mRNA) is inherently unstable and prone to degradation by nucleases and self-hydrolysis. Encapsulation of mRNA within LNPs protects the mRNA from extracellular ribonucleases and assists with intracellular mRNA delivery.

[0011] A variety of lipids have been developed for use in LNPs, yet there remains a need for specific formulations for nucleic acid delivery, especially complicated nucleic acid cargoes involving mRNA, that can effectively be used in specific tissues or organs (e.g., retina).

[0012] SUMMARY

[0013] Accordingly, the present disclosure describes, in part, various lipid nanoparticles, e.g., for delivering one or more nucleic acid-based agents to the retina.

[0014] In aspects, the present compositions provide one or more lipids in association with one or more nucleic acidbased agents. In embodiments the one or more lipids associate into an aggregate. In embodiments the one or more lipids associate into a lipid nanoparticle (LNP). In embodiments the one or more lipids, e.g., LNP, encapsulate the one or more nucleic acid-based agents. In embodiments the LNP comprising the one or more nucleic acid-based agents is suitable for delivery of the one or more nucleic acid-based agents in a subject. In embodiments the LNP comprising the one or more nucleic acid-based agents is formulated with a pharmaceutically acceptable carrier or excipient.

[0015] In embodiments, the nucleic acid-based agent comprises both a nucleic acid encoding a mobile element enzyme in the form of modified mRNA (mmRNA) and a donor DNA or transgene or mobile element in the form of plasmid DNA. In embodiments, the weight to weight ratio of plasmid DNA : mmRNA is about 5 : about 1 , or about 2 : about 1 , or about 1 : about 1 , or about 1 : about 2, or about 1 : about 5.

[0016] In aspects, there is provided a method for introducing a nucleic acid-based agent into a cell, the method comprising contacting the cell with a composition described herein.

[0017] In aspects, there is provided a method for in vivo delivery of a nucleic acid-based agent, the method comprising administering to a subject a composition described herein.

[0018] In aspects, there is provided a method for ocular delivery of nucleic acid, the method comprising ocularly administering to a subject a composition described herein.

[0019] In aspects, there is provided a method for treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition described herein.

[0020] In aspects, there is provided a method for treating an ocular disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition described herein.

[0021] BRIEF DESCRIPTION OF DRAWINGS

[0022] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0023] FIG. 1 shows encapsulation efficiencies of compositions of the disclosure with components of the same.

[0024] FIG. 2 shows formulations of MC3 and C12-200 mixed LNPs and physical properties thereof.

[0025] FIG. 3A and FIG. 3B show alteration of the amount of PEG in compositions. For each condition in FIG. 3A, the left bar refers to size and the right bar refers to polydispersity index (PDI). FIG. 4A and FIG. 4B show the effects of different PEG identities and conjugated lipid length. For each condition in FIG. 4A, the left bar refers to size and the right bar refers to PDI.

[0026] FIG. 5A and FIG. 5B show the effects of different pHs on %EE. For each condition in FIG. 5A, the left bar refers to size and the right bar refers to PDI.

[0027] FIG. 6 shows SS-OP LNPs and physical properties thereof.

[0028] FIG. 7 shows the compositions of multiple formulations that can be injected into mice sub-retinally.

[0029] FIG. 8 shows the effects of altering the amount of Cationic / lonizable lipid in formulations of the disclosure.

[0030] FIG. 9 shows LNPs screening with a method suitable for use in certain organs or tissues (e.g, or the retina).

[0031] FIG. 10 shows the selection of beneficial formulations for mRNA delivery with a luciferase reporter.

[0032] FIG. 11 shows a non-limiting study design study design for various formulations of the disclosure (Ionizable lipids and hydrolysable lipids) to be injected sub-retinally.

[0033] FIG. 12 is a series of fundus and GFP images demonstrating LNP tolerability and expression with various SS-OP, and DOTAP / PEG formulations of the disclosure.

[0034] FIG. 13 is a series of optical coherence tomography (OCT) images and a histogram demonstrating the level of cell death and tolerability with various SS-OP, and DOTAP / PEG of the disclosure. For each condition, the bars right to left refer to D1 , D7, D14 and D21.

[0035] FIG. 14 is a dot plot depicting that SS-OP and DOTAP / PEG formulations of the disclosure of LNPs provide high level of mRNA expression with low toxicity. For each condition, left points refer to eyecup / RPE and right points refer to retina.

[0036] FIG. 15 shows the study design for various formulations (ionizable lipids and hydrolyzable lipids) for sub- retinal injection.

[0037] FIG. 16 is a series of fundus and GFP images demonstrating LNP tolerability and expression with various SS-OP of the disclosure.

[0038] FIG. 17 shows a series of fundus and Fluorescent GFP images demonstrating LNP tolerability and expression with SS_EC and DOTAP formulations of the disclosure.

[0039] FIG. 18 is a series of optical coherence tomography (OCT) images demonstrating level of cell death and tolerability with various SS-OP, SS-EC, and DOTAP / PEG formulations of the disclosure. FIG. 19 shows a histogram demonstrating the level of cell death and tolerability with histograms demonstrating level of cell death and tolerability with various SS-OP, SS-EC, and DOTAP / PEG formulations of the disclosure. For each condition, the bars right to left refer to D1 , D7, D14 and D21.

[0040] FIGS. 20A-20B show screening of LNPs with immuno-silent mRNA cargo (GFP) to select lipids with a favourable tolerability profile in vivo. FIG. 20A: Tolerability was evaluated by optical coherence tomography (OCT) imaging of retina in-life (at 3 days post injection) which includes the treated area and the opposite side (non-injected). Thickness of the outer nuclear layer (ONL) which contains photoreceptors indicates tolerability of the LNP formulation. As photoreceptor cell death occurs, the ONL thins, which is qualitatively demonstrated via the scoring system. A score of 0 represents <10% loss of thickness in ONL compared to the non-injected region; score of 1 represents 10-50% loss of ONL thickness compared to the non-injected region; and score of 2 represents >50% loss of ONL thickness in the treated area compared to the non-injected region. FIG. 20B: Disclosed LNPs showed minimal degeneration compared to standard LNPs. LNP 1: SS-OP (13:1 SS- OP:NA, 1 :37:5.3 %mol DOPE:% mol SS-OP: %mol Cholesterol; 5% PEG); LNP 2: SSEC (13:1 SSEC:NA, 1 :1 :2 %mol DOPE:% mol SSEC: %mol Cholesterol; 5% PEG); Std LNP: ALC-0315 (10:1 ALC- 0315:NA, 1 :1 :4 %mol DOPE:% mol ALC-0315: %mol Cholesterol; 2.5% PEG). Graphing the ONL degeneration from the study indicates that the SS-OP formulations containing GFP mRNA only are best tolerated in vivo compared to SSEC and ALC-0315 formulations. The degeneration score indicated that lipids similar to ALC-0315 are not tolerated for mRNA and thus are not of interest for DNA; however, the use of SS-OP formulation is suitable for dual cargo delivery.

[0041] FIGS. 21 A-21 B are series of images and graphs showing that subretinal (SR) injection of SS-OP / mRNA was delivered to neural retina and RPE. SS-OP / mRNA was delivered to -60% of mouse neural retina cells within the treated area. The neural retina is composed of many cell types, including photoreceptors (PRs). Subretinal injection of Cre reporter mRNA SS-OP LNPs (36.3% SS-OP, 50.7% Cholesterol, 9.8% DOPE, 5% PEG) in mice was quantified using immunohistochemistry. FIG. 21 A: Images illustrating the back of the retina of which 30% is the treated area (area which received the 1 uL injection). Cre expression was limited to the treated area indicating substantial delivery of SS-OP LNPs into the retina. These images compared the Vehicle (PBS) showing no Cre expression in the retinal ganglion cells (RGC) inner and outer nuclear layers (INL, ONL), to the SS-OP-Cre mRNA LNP. LNP stained with red color indicates delivery and expression of mRNA into all 3 cell types, of which INL and ONL contain the photoreceptors. DAPI staining stains the nucleus of the cells blue and allows for the overlay to demonstrate Cre expression in the photoreceptor cell body and the cell segments represented by the hexagonal pattern staining. FIG. 21 B: The graph shows % neural retina cells transfected with the SS-OP LNP of which 80% are photoreceptors. A dose response is seen, where 0.2 ug / eye of Cre reporter mRNA shows the highest % transfection (-60%) compared to the other doses. A decrease in expression could be caused, without wishing to be bound by theory, by low tolerability at higher doses. The LNP here was formulated using a Precision Nanosystems Ignite system with the size -80 nm and PDI < 0.2.

[0042] FIG. 22 is a series of images demonstrating that SS-OP / mRNA were delivered into nonhuman primates (NHP) photoreceptors (PRs) and retinal pigment epithelial (RPE) cells NHP tolerability was comparable to vehicle. Subretinal (SR) injection of GFP reporter mRNA SS-OP LNPs (36.3% SS-OP, 50.7% Cholesterol, 9.8% DOPE, 5% PEG) was performed in non-human primates. The formulations were made in aseptic conditions with 75 nm in size and <0.2 PDI. 24 hrs post-injections, the NHPs were euthanized and eyes were collected for IHC. The top image panel shows the interface between the treated and the non-treated area. DAPI stains the nuclei in blue and GFP is stained in green. As shown, PRs comprised the outer nuclear layer (ONL) and Inner and Outer segments with retinal pigment epithelial cells (RPEs) at the bottom. Green GFP expression was detected in a majority of the cell population including the photoreceptors, within the treated area. The expression decreased to zero outside the treated area, representative of LNP-based expression in vivo. The bottom panels are zoomed in versions of the top panel indicating that GFP expression was detected within the photoreceptor IS, OS and the ONL. The dotted, hexagonal pattern represents the photoreceptor cell structure, where GFP expression was detected.

[0043] FIG. 23 is a histogram demonstrating the therapeutic window for SS-OP / ABCA4 DNA delivery and that tolerability is driven by the copy number of DNA delivered. GFP vs. Abca4 were used as cargo within SS-OP LNPs (36.3% SS-OP, 50.7% Cholesterol, 9.8% DOPE, 5% PEG) and the therapeutic index was evaluated. OCT measurements were taken at day 12 (D12) of the mice injected subretinally with the LNPs. The degeneration score indicated that tolerability is driven by the number of molecules of DNA delivery, regardless of the DNA size or sequence. The x axis provides the nucleic acid concentration in nM and corresponding abca4 and GFP amounts in ng. Based on the differences in cargo base pair size, the amount of abca4 or GFP was modified to match the same nM of nucleic acid. Here, 7 nM was shown to be the therapeutic index for the DNA regardless of the cargo size with a OCT degeneration score <1 on average. For orientation purposes, the third, fifth, and sixth bars represent ABCA4, and the fourth, seventh and eighth bars represent GFP DNA. FIGS. 24A-24B are series of histograms depicting that SS-OP / ABCA4 delivery to photoreceptors (PRs) and retinal pigment epithelial (RPE) cells following subretinal (SR) injection was within current therapeutic index (Tl). FIG. 24A: Disclosed formulations deliver preferentially to the RPE, with lower efficiency in the neural retina. Quantifying DNA delivery and the transcript produced. For 7 nM of DNA (25 ng for GFP and 50 ng for Abca4) Copy number per cell are graphed. 0.01 copies / cell for neural retina and 1 copy / cell for RPE are seen for both Abca4 and GFP. Overall, abca4 DNA delivery is lowered compared to GFP. For orientation purposes, in each condition, the bars on the left represent GFP DNA and the bars on the right represent ABCA4 DNA. FIG. 24B: Transcription of the DNA into mRNA by using RT-qPCR fold change compared to PBS blank. Both Abca4 and GFP have the same level of fold change indicating increased levels of mRNA produced from the DNA, indicating that LNP delivery of Abca4 is productive and leads to transcription of mRNA as well. For orientation purposes, in each condition, the bars on the left represent GFP DNA and the bars on the right represent ABCA4 DNA.

[0044] FIGS. 25A-25B are series of images and a graph showing neural retina integration following subretinal (SR) delivery of AAV5 with LNP and the present mobile element enzyme (SEQ ID NO: 1). The study evaluated dual subretinal delivery into mice staggered by 21 days (AAV followed by LNP with Cre mRNA-HMobile element enzyme (SEQ ID NO: 1) mRNA (36.3% SS-OP, 50.7% Cholesterol, 9.8% DOPE, 5% PEG)). FIG. 25A: fundus images taken in the Green GFP channel indicating GFP expression of AAV5 and red-tdTomato channel indicating widespread expression by the Cre LNP. In the dual injected groups, the merge demonstrated a large population of cells received dual mRNA leading to dual protein expression. It is concluded that cells that received Cre mRNA and showed a positive expression, also received the present mobile element enzyme (SEQ ID NO: 1) mRNA given that they were coencapsulated in the same LNP. FIG. 25B: The copy number per cell via ddPCR of cells in the neural retina (-80% photoreceptors) that express both GFP from AAV and cre / mobile element enzyme (SEQ ID NO: 1) from LNP. All doses, with 0.05 ug / ml being the highest show an increase in DNA copy number compared to AAV alone, indicative of integration via Mobile element enzyme (SEQ ID NO: 1). Increased levels of mobile element enzyme (SEQ ID NO: 1) showed low tolerability, as demonstrated by a decrease in DNA copy number.

[0045] FIGS. 26A-26B are series of images and a graph showing that ABCA4 + mobile element enzyme (SEQ ID NO: 1) mRNA delivery into photoreceptors (PRs) occurred with good tolerability. The formulation evaluated was SS-OP LNP (36.3% SS-OP, 50.7% Cholesterol, 9.8% DOPE, 5% PEG)) with CAG-hAbca4 NP and mobile element enzyme (SEQ ID NO: 1) mRNA. FIG. 26A: Minimal to a no degeneration was observed with SS-OP / ABCA4+ mobile element enzyme (SEQ ID NO: 1) delivery to the retina. The OCT degeneration score of the ONL is within the tolerable range for LNPs for both doses. An average score of < 0.5 indicates high tolerability for this dual loaded LNP. For orientation purposes, in each condition, Day 9 is on the left and Day 14 is on the right. FIG. 26B: ABCA4 expression corresponds to about 5-10% of photoreceptors (PRs) in the entire retina FACS showing data isolating photoreceptors (CD73+) from the whole retina and further isolating for Abca4+ cells. Compared to background (2.7%) both doses of LNP indicate increased Abca4 expression of > 5%. Abca4 protein is therefore expressed in 5-10% of the Photoreceptors in the neural retina whereas only 10-15% of Abca4+ cells are needed in the treated area. Note: treated area is 1 / 3 of whole retina.

[0046] FIG. 27 is a series of images demonstrating that SS-OP / ABCA4 and mobile element enzyme (SEQ ID NO: 1) are expressed in mouse photoreceptors (PRs). Immunohistochemistry (IHC) assays showed DAPI stained, PNA lectin stains the inner and outer segments of the photoreceptors, RPE65 stains the RPE layer, and Abca4 is labelled. As expected, the Abca4 was localized to the outer segments of the photoreceptors which themselves were naturally expressed as a transport protein. The localization showed that the LNP was able to deliver the Abca4 DNA and the DNA was able to be translated into protein that is expressed in the OS of the photoreceptors.

[0047] DETAILED DESCRIPTION

[0048] In aspects, the present compositions (also referred to as “formulations” herein) provide one or more lipids in association with one or more nucleic acid-based agents. In embodiments the one or more lipids associate into an aggregate. In embodiments the one or more lipids associate into a lipid nanoparticle (LNP). In embodiments, the one or more lipids, e.g., LNP, encapsulate the one or more nucleic acid-based agents. In embodiments, the composition or LNP comprising the one or more nucleic acid-based agents is suitable for delivery of the one or more nucleic acid-based agents in a subject. In embodiments, the composition or LNP comprising the one or more nucleic acid-based agents is formulated with a pharmaceutically acceptable carrier or excipient.

[0049] Lipids

[0050] In embodiments, the one or more lipids compact the one or more nucleic acid-based agents to be delivered.

[0051] In embodiments, the one or more lipids protect the one or more nucleic acid-based agents to be delivered from nuclease degradation.

[0052] In embodiments, the one or more lipids cause, promote or enhance receptor-mediated uptake of the one or more nucleic acid-based agents, e.g, by a cell. In embodiments, the one or more lipids, e.g., when contacted with a cell, cause, promote or enhance association with negatively charged cellular membranes.

[0053] In embodiments, the one or more lipids, e.g., when contacted with a cell, cause, promote or enhance fusion with endosomal membranes, without wishing to be bound by theory, facilitating the release of complexes from endosomal compartments.

[0054] In embodiments, the one or more lipids, e.g., when contacted with a cell, cause, promote or enhance transport of the one or more nucleic acid-based agents from the cytoplasm to the nucleus.

[0055] In embodiments, the one or more lipids comprise cationic lipids; anionic lipids; neutral lipids; multi-valent charged lipids; and zwitterionic lipids.

[0056] In embodiments, the one or more lipids include a delivery lipid. In embodiments, the delivery lipid is or comprises a cationic lipid. In embodiments, the cationic lipid is ionizable and / or hydrolysable. In embodiments, the delivery lipid encapsulates, in whole or in part, the one or more nucleic acid-based agents. In embodiments, the delivery lipid, e.g., when contacted with a cell, promotes or increases endosomal escape. In embodiments, the delivery lipid is or comprises MC3 (DLin-MC3-DMA), C12-200 (1, T-((2-(4-(2- ((2-(bis(2-hydroxydodecyl)amino)ethyl) (2-hydroxydodecyl)amino)ethyl)piperazin-1- yl)ethyl)azanediyl)bis(dodecan-2-ol)), SM102 (9-Heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6- (undecyloxy)hexyl]amino}octanoate), squaramide (3, 4-diaminocyclobut-3-ene-1, 2-dione; SQAM), SS-EC (ssPalmE-P4C2), SS-OP (ssPalmO-Phe), [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2- hexyl decanoate), KC2 (DLin-KC2-DMA), DOTAP (Dioleoyl-3-trimethylammonium propane), cKK-E12 (3,6- bis({4-[bis(2-hydroxydodecyl)amino]butyl})piperazine-2, 5-dione), ATX-001 (di((Z)-non-2-en-1-yl) 8,8'-((2-((2- (dimethylamino)ethyl)thio)acetyl)azanediyl)dioctanoate), ATX-002 (di((Z)-non-2-en-1-yl) 8,8'-((((2-

[0057] (dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate), ATX-012, ATX-61, ATX-0081 , ATX-0095, Lipid 5 (8-[(2-hydroxyethyl)[8-(nonyloxy)-8-oxooctyl]amino]-octanoic acid, 1 -octylnonyl ester), and / or ALC-0315 (2- hexyl-decanoic acid, 1 ,1 '-[[(4-hydroxybutyl)imino]di-6,1 -hexanediyl] ester, [(4- Hydroxybutyl)azanediyl]di(hexane-6,1 -diyl) bis(2-hexyldecanoate)). Various ATX lipids that are in embodiments delivery lipids of the disclosure are described in WO2018222890 and WO2015074085, each of which is hearby incorporate by reference in its entirety. In embodiments, the delivery lipid is or comprises DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, DLin-MC3-DMA, 98N12-5, and C12-200. In embodiments, the one or more lipids include one type of delivery lipid. In embodiments, the one or more lipids include more than one type of delivery lipid. In embodiments, the one or more lipids include two types of delivery lipid. In embodiments, the one or more lipids include both MC3 and C12-200.

[0058] In embodiments, the one or more lipids include cholesterol. In embodiments, the cholesterol provides increased or enhanced LNP stability.

[0059] In embodiments, the one or more lipids include a helper lipid. In embodiments, the helper lipid encapsulates, in whole or in part, the one or more nucleic acid-based agents. In embodiments, the helper lipid, e.g., when contacted with a cell, promotes or increases endosomal escape. In embodiments, the helper lipid is a phospholipid. In embodiments the helper lipid is a neutral lipid. In embodiments, the neutral lipid is or comprises dioleoylphosphatidylethanolamine (DOPE) and / or 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC). In embodiments, the helper lipid is a cationic lipid. In embodiments, the cationic lipid is or comprises N-[1 - (2, 3-d ioleoyloxy) p ropy I]- N, N, N-trimethylammonium chloride (DOTMA), 1 ,2-bis(oleoyloxy)-3-3- (trimethylammonia) propane (DOTAP), or 1 ,2-dioleoyl-3-dimethylammonium-propane (DODAP). In embodiments, the helper lipid is or comprises DOPE, DOPC, and / or Distearoylphosphatidylcholine (DSPC). In embodiments, the helper lipid is or comprises both DOPE and DSPC. In embodiments, the helper lipid is or comprises both DOPE and DOPC. In embodiments, the helper lipid is or comprises all of DOPE, DSPC, and DOPC.

[0060] In embodiments, the one or more lipids include a PEGylated lipid. In embodiments, the PEGylated lipid shields the LNP and reduces or prevents degradation of the LNP, e.g., in the bloodstream. In embodiments, the PEGylated lipid has a PEG molecule covalently attached to it, where the PEG has an average molecular weight of from about 1 kDa to about 50 kDa, or about 1 kDa to about 30 kDa, or from about 1 kDa to about 10 kDa, or about 1 kDa, or about 2 kDa, or about 3 kDa, or about 4 kDa, or about 5 kDa, or about 10 kDa. In embodiments, the PEG is a linear, a branched PEG, a star PEG, or a comb PEG. In embodiments the PEG is selected from PEG200, PEG300, PEG400, PEG600, PEG800, PEG1000, PEG1500, PEG2000, PEG3000, and PEG4000. In embodiments, the PEG is PEG2000. In embodiments the PEGylated lipid is or comprises 1 ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG). In embodiments, the DMG- PEG is or comprises 1 , 2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-2k- DMG) and / or a PEG conjugated to a ceramide, e.g., C8 PEG 2k Ceramide, C16 PEG 2k Ceramide. In embodiments the PEGylated lipid is or comprises PEG-dimyristyloxypropyl (PEG-DMA), a PEG-distearyloxypropyl (PEG-DSA), PEG-diacylglycerol (PEG-DAG), or PEG-dialkyloxypropyl (PEG-DAA). In embodiments, the one or more lipids do not include a PEGylated lipid. In embodiments, the one or more lipids do not include a PEGylated lipid, e.g., when being formulated for local administration.

[0061] In embodiments, the composition comprises a mixture of PEGylated lipids and free PEG chains.

[0062] Compositions / Formulations

[0063] In embodiments, there is provided a composition comprising:

[0064] (a) one or more nucleic acid-based agents, and

[0065] (b) one or more lipids, the one or more lipids comprising:

[0066] (i) a delivery lipid,

[0067] (ii) cholesterol,

[0068] (iii) a helper lipid, and

[0069] (iv) a PEGylated lipid.

[0070] In embodiments, the delivery lipid is selected from MC3, SS-OP, SS_EC, and any combination thereof.

[0071] In embodiments, the helper lipid is selected from distearoylphosphatidylcholine (DSPC), 1,2-Dioleoyl-sn- glycero-3-phosphocholine (DOPC), and dioleoylphosphatidylethanolamine (DOPE).

[0072] In embodiments, the PEGylated lipid is selected from PEG-2K-DMG, C8 PEG 2K, and C16 PEG 2K. In embodiments, the one or more lipids do not comprise a PEGylated lipid.

[0073] In embodiments, the composition has a weight to weight ratio of delivery lipid to one or more nucleic acidbased agents of about 7:1 , or about 8:1 , or about 9:1 , or about 10:1 , or about 11 :1 , or about 12:1, or about 13:1 , and / or a has a weight to weight ratio of total lipid to one or more nucleic acid-based agents of about 14: 1 , or about 15: 1 , or about 16: 1 , or about 17: 1 , or about 18: 1 , or about 19: 1 , or about 20:1 , or about 21:1 , or about 22: 1 , or about 23: 1 , or about 24: 1 , or about 25: 1 , or about 26:1.

[0074] In embodiments, the composition has a weight to weight ratio of delivery lipid to one or more nucleic acidbased agents from about 7:1 to about 13:1 and / or a has a weight to weight ratio of total lipid to one or more nucleic acid-based agents from about 14:1 to about 26:1 .

[0075] In embodiments, the composition has a weight to weight ratio of delivery lipid to one or more nucleic acidbased agents of about 7:1 and / or a has a weight to weight ratio of total lipid to one or more nucleic acidbased agents of about 25:1. In embodiments, the

[0076] (i) a delivery lipid comprising from about 15 mol % to about 75 mol % of the total lipid present,

[0077] (ii) cholesterol comprising from about 25 mol % to about 65 mol % of the total lipid present,

[0078] (iii) a helper lipid comprising from about 5 mol % to about 40 mol % of the total lipid present, and

[0079] (iv) a PEGylated lipid comprising from about 0.5 mol % to about 7 mol % of the total lipid present.

[0080] In embodiments, the

[0081] (i) a delivery lipid comprising from about 25 mol % to about 75 mol % of the total lipid present,

[0082] (ii) cholesterol comprising from about 25 mol % to about 65 mol % of the total lipid present,

[0083] (iii) a helper lipid comprising from about 5 mol % to about 40 mol % of the total lipid present, and

[0084] (iv) a PEGylated lipid comprising from about 0.5 mol % to about 3 mol % of the total lipid present.

[0085] In embodiments, the one or more lipids comprises:

[0086] (i) a delivery lipid comprising from about 30 mol % to about 60 mol % of the total lipid present, the delivery lipid being MC3,

[0087] (ii) cholesterol comprising from about 30 mol % to about 60 mol % of the total lipid present,

[0088] (iii) a helper lipid comprising from about 5 mol % to about 35 mol % of the total lipid present, optionally being one or more of:

[0089] DSPC from about 8 mol % to about 12 mol % of the total lipid present,

[0090] DOPC from about 8 mol % to about 10 mol % of the total lipid present, and

[0091] DOPE from about 7.5 mol % to about 25 mol % of the total lipid present, and

[0092] (iv) a PEGylated lipid comprising from about 0.5 mol % to about 3 mol % of the total lipid present, optionally being one or more of:

[0093] PEG-2K-DMG from about about 0.5 mol % to about 3 mol % of the total lipid present,

[0094] C8 PEG 2K ceramide from about about 0.5 mol % to about 2.5 mol % of the total lipid present, and C16 PEG 2K ceramide from about about 0.5 mol % to about 2.5 mol % of the total lipid present.

[0095] In embodiments, the one or more lipids comprises: In embodiments, the one or more lipids comprises:

[0096] (i) a delivery lipid comprising about 45 mol % of the total lipid present, the delivery lipid being MC3,

[0097] (ii) cholesterol comprising about 45 mol % of the total lipid present,

[0098] (iii) a helper lipid comprising about 10 mol % of the total lipid present, the helper lipid being DOPE, and (iv) a PEGylated lipid comprising about 2 mol % of the total lipid present, the PEGylated lipid being PEG-2K-DMG.

[0099] In embodiments, the one or more lipids comprises:

[0100] (i) a delivery lipid comprising about 45 mol % of the total lipid present, the delivery lipid being MC3,

[0101] (ii) cholesterol comprising about 45 mol % of the total lipid present, (iii) a helper lipid comprising about 10 mol % of the total lipid present, the helper lipid being DOPE, and

[0102] (iv) a PEGylated lipid comprising about 2 mol % of the total lipid present, the PEGylated lipid being PEG-2K-DMG and a nucleic acid-based agent having a weight to weight ratio of delivery lipid to one or more nucleic acid-based agents of about 13: 1 and / or a having a weight to weight ratio of total lipid to one or more nucleic acid-based agents of about 25:1 .

[0103] In embodiments, the one or more lipids comprises:

[0104] (i) a delivery lipid comprising from about 25 mol % to about 75 mol % of the total lipid present, the delivery lipid being SS-OP,

[0105] (ii) cholesterol comprising from about 25 mol % to about 65 mol % of the total lipid present,

[0106] (iii) a helper lipid comprising from about 5 mol % to about 30 mol % of the total lipid present, optionally being one or more of:

[0107] DOPC from about 5 mol % to about 30 mol % of the total lipid present, and

[0108] DOPE from about 2.5 mol % to about 30 mol % of the total lipid present, and

[0109] (iv) a PEGylated lipid comprising from about 0.5 mol % to about 3 mol % of the total lipid present, optionally being PEG-2K-DMG.

[0110] In embodiments, the one or more lipids comprises:

[0111] In embodiments, the one or more lipids comprises:

[0112] (i) a delivery lipid comprising about 15 mol %, about 16 mol%, about 17 mol%, about 18 mol%, about

[0113] 19 mol%, about 20 mol%, about 21 mol%, about 22 mol%, about 23 mol%, about 24 mol%, about

[0114] 25 mol%, about 26 mol%, about 27 mol%, about 28 mol%, about 29 mol%, about 30 mol%, about

[0115] 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about

[0116] 37 mol%, about 38 mol%, about 39 mol%, about 40 mol%, about 41 mol%, about 42 mol%, about

[0117] 43 mol%, about 44 mol%, or about 45 mol % of the total lipid present, the delivery lipid being SS- OP,

[0118] (ii) cholesterol comprising about 35 mol %, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, 45 mol %, about 46 mol%, about 47 mol%, about 48 mol%, about 49 mol%, about 50 mol%, about 51 mol%, about 52 mol%, about 53 mol%, about 54 mol%, about 55 mol %, about 56 mol%, about 57 mol%, about 58 mol%, about 59 mol%, or about 60 mol % of the total lipid present,

[0119] (iii) a helper lipid comprising about 5 mol %, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, about

[0120] 15 mol %, about 16 mol%, about 17 mol%, about 18 mol%, about 19 mol%, about 20 mol%, about

[0121] 21 mol%, about 22 mol%, about 23 mol%, about 24 mol%, about 25 mol%, about 26 mol%, about

[0122] 27 mol%, about 28 mol%, about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about

[0123] 33 mol%, about 34 mol%, or about 35 mol % of the total lipid present, optionally being one or more of DOPE or DSPC, and

[0124] (iv) a PEGylated lipid comprising from about 3 mol %, about 4 mol%, about 5 mol%, about 6 mol%, or about 7 mol % of the total lipid present, optionally being PEG-2K-DMG.

[0125] In embodiments, the one or more lipids comprises:

[0126] (i) a delivery lipid comprising from about 15 mol % to about 45 mol % of the total lipid present, the delivery lipid being SS-OP,

[0127] (ii) cholesterol comprising from about 35 mol % to about 60 mol % of the total lipid present, (iii) a helper lipid comprising from about 5 mol % to about 35 mol % of the total lipid present, optionally being one or more of DOPE or DSPC, and

[0128] (iv) a PEGylated lipid comprising from about 3 mol % to about 7 mol % of the total lipid present, optionally being PEG-2K-DMG.

[0129] In embodiments, the one or more lipids comprises:

[0130] (i) a delivery lipid comprising from about 22 mol % to about 39 mol % of the total lipid present, the delivery lipid being SS-OP,

[0131] (ii) cholesterol comprising from about 46 mol % to about 53 mol % of the total lipid present,

[0132] (iii) a helper lipid comprising from about 7 mol % to about 26 mol % of the total lipid present, optionally being one or more of DOPE or DSPC, and

[0133] (iv) a PEGylated lipid comprising from about 4.5 mol % to about 5.5 mol % of the total lipid present, optionally being PEG-2K-DMG.

[0134] In embodiments, the one or more lipids comprises:

[0135] (i) a delivery lipid comprising from about 22 mol % to about 26 mol % of the total lipid present, the delivery lipid being SS-OP,

[0136] (ii) cholesterol comprising from about 46 mol % to about 50 mol % of the total lipid present,

[0137] (iii) a helper lipid comprising from about 22 mol % to about 26 mol % of the total lipid present, optionally being one or more of DOPE or DSPC, and

[0138] (iv) a PEGylated lipid comprising from about 4 mol % to about 6 mol % of the total lipid present, optionally being PEG-2K-DMG.

[0139] In embodiments, the one or more lipids comprises:

[0140] (i) a delivery lipid comprising from about 34 mol % to about 38 mol % of the total lipid present, the delivery lipid being SS-OP,

[0141] (ii) cholesterol comprising from about 48 mol % to about 52 mol % of the total lipid present,

[0142] (iii) a helper lipid comprising from about 8 mol % to about 10 mol % of the total lipid present, optionally being one or more of DOPE or DSPC, and (iv) a PEGylated lipid comprising from about 4 mol % to about 6 mol % of the total lipid present, optionally being PEG-2K-DMG.

[0143] In embodiments, the one or more lipids comprises:

[0144] (i) a delivery lipid comprising about 36 mol % of the total lipid present, the delivery lipid being SS- OP,

[0145] (ii) cholesterol comprising about 51 mol % of the total lipid present,

[0146] (iii) a helper lipid comprising about 10 mol % of the total lipid present, optionally being one or more of DOPE or DSPC, and

[0147] (iv) a PEGylated lipid comprising about 5 mol % of the total lipid present, optionally being PEG-2K- DMG.

[0148] In embodiments, the one or more lipids comprises:

[0149] (i) a delivery lipid comprising about 36 mol % of the total lipid present, the delivery lipid being SS- OP,

[0150] (ii) cholesterol comprising about 51 mol % of the total lipid present,

[0151] (iii) a helper lipid comprising about 10 mol % of the total lipid present, optionally being one or more of DOPE or DSPC, and

[0152] (iv) a PEGylated lipid comprising about 5 mol % of the total lipid present, optionally being PEG-2K- DMG, and a nucleic acid-based agent having a weight to weight ratio of delivery lipid to one or more nucleic acid-based agents of about 7:1 and / or a having a weight to weight ratio of total lipid to one or more nucleic acid-based agents of about 25:1 .

[0153] In embodiments, the one or more lipids comprises:

[0154] (i) a delivery lipid comprising about 24 mol % of the total lipid present, the delivery lipid being SS- OP,

[0155] (ii) cholesterol comprising about 48 mol % of the total lipid present,

[0156] (iii) a helper lipid comprising about 24 mol % of the total lipid present, optionally being one or more of DOPE or DSPC, and (iv) a PEGylated lipid comprising about 5 mol % of the total lipid present, optionally being PEG-2K- DMG.

[0157] In embodiments, the one or more lipids comprises:

[0158] (i) a delivery lipid comprising about 24 mol % of the total lipid present, the delivery lipid being SS- OP,

[0159] (ii) cholesterol comprising about 48 mol % of the total lipid present,

[0160] (iii) a helper lipid comprising about 24 mol % of the total lipid present, optionally being one or more of DOPE or DSPC, and

[0161] (iv) a PEGylated lipid comprising about 5 mol % of the total lipid present, optionally being PEG-2K- DMG, and a nucleic acid-based agent having a weight to weight ratio of delivery lipid to one or more nucleic acid-based agents of about 7:1 and / or a having a weight to weight ratio of total lipid to one or more nucleic acid-based agents of about 25:1 .

[0162] In embodiments, the one or more lipids comprises:

[0163] In embodiments, the one or more lipids comprises:

[0164] (i) a delivery lipid comprising about 15 mol %, about 16 mol%, about 17 mol%, about 18 mol%, about 19 mol%, about 20 mol%, about 21 mol%, about 22 mol%, about 23 mol%, about 24 mol%, about 25 mol%, about 26 mol%, about 27 mol%, about 28 mol%, about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, or about 35 mol% of the total lipid present, the delivery lipid being SS-EC,

[0165] (ii) cholesterol comprising about 35 mol %, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, 45 mol %, about 46 mol%, about 47 mol%, about 48 mol%, about 49 mol%, about 50 mol%, about 51 mol%, about 52 mol%, about 53 mol%, about 54 mol%, about 55 mol %, about 56 mol%, about 57 mol%, about 58 mol%, about 59 mol%, or about 60 mol % of the total lipid present,

[0166] (iii) a helper lipid comprising about 15 mol %, about 16 mol%, about 17 mol%, about 18 mol%, about 19 mol%, about 20 mol%, about 21 mol%, about 22 mol%, about 23 mol%, about 24 mol%, about 25 mol%, about 26 mol%, about 27 mol%, about 28 mol%, about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, or about 35 mol % of the total lipid present, optionally being one or more of DOPE or DSPC, and

[0167] (iv) a PEGylated lipid comprising from about 3 mol %, about 4 mol%, about 5 mol%, about 6 mol%, or about 7 mol % of the total lipid present, optionally being PEG-2K-DMG.

[0168] In embodiments, the one or more lipids comprises:

[0169] (i) a delivery lipid comprising from about 15 mol % to about 35 mol % of the total lipid present, the delivery lipid being SS-EC,

[0170] (ii) cholesterol comprising from about 35 mol % to about 60 mol % of the total lipid present,

[0171] (iii) a helper lipid comprising from about 15 mol % to about 35 mol % of the total lipid present, optionally being one or more of DOPE or DSPC, and

[0172] (iv) a PEGylated lipid comprising from about 3 mol % to about 7 mol % of the total lipid present, optionally being PEG-2K-DMG.

[0173] In embodiments, the one or more lipids comprises:

[0174] (i) a delivery lipid comprising from about 22 mol % to about 26 mol % of the total lipid present, the delivery lipid being SS-EC,

[0175] (ii) cholesterol comprising from about 46 mol % to about 50 mol % of the total lipid present,

[0176] (iii) a helper lipid comprising from about 22 mol % to about 26 mol % of the total lipid present, optionally being one or more of DOPE or DSPC, and

[0177] (iv) a PEGylated lipid comprising from about 4.5 mol % to about 5.5 mol % of the total lipid present, optionally being PEG-2K-DMG.

[0178] In embodiments, the one or more lipids comprises:

[0179] (i) a delivery lipid comprising about 24 mol % of the total lipid present, the delivery lipid being SS- EC, (ii) cholesterol comprising about 48 mol % of the total lipid present,

[0180] (iii) a helper lipid comprising about 24 mol % of the total lipid present, optionally being one or more of DOPE or DSPC, and

[0181] (iv) a PEGylated lipid comprising about 5 mol % of the total lipid present, optionally being PEG-2K- DMG.

[0182] In embodiments, the one or more lipids comprises:

[0183] (i) a delivery lipid comprising about 24 mol % of the total lipid present, the delivery lipid being SS- EC,

[0184] (ii) cholesterol comprising about 48 mol % of the total lipid present,

[0185] (iii) a helper lipid comprising about 24 mol % of the total lipid present, optionally being one or more of DOPE or DSPC, and

[0186] (iv) a PEGylated lipid comprising about 5 mol % of the total lipid present, optionally being PEG-2K- DMG, and a nucleic acid-based agent having a weight to weight ratio of delivery lipid to one or more nucleic acid-based agents of about 7:1 and / or a having a weight to weight ratio of total lipid to one or more nucleic acid-based agents of about 25:1 .

[0187] In embodiments, the composition comprises one of: F5, and F6 of FIG. 1.

[0188] In embodiments, the composition comprises one of: F1 , and F8 of FIG. 2.

[0189] In embodiments, the composition comprises one of the formulations of FIG. 3A and FIG. 3B.

[0190] In embodiments, the composition comprises one of the formulations of FIG. 4A and FIG. 4B.

[0191] In embodiments, the composition comprises one of the formulations of FIG. 5A and FIG. 5B.

[0192] In embodiments, the composition comprises one of the formulations of FIG. 6.

[0193] In embodiments, the composition comprises one of the formulations of FIG. 7.

[0194] In embodiments, the composition comprises one of the formulations: F1, F2, F3, F4, F5, and F6 of FIG. 8.

[0195] In embodiments, the composition comprises one of the formulations: F1 , F2, F3, F4, F5, F6, and F7 of FIG.

[0196] 9. In embodiments, the composition comprises one of the formulations from the groups: 2, 3, 4, 5, 6, 7, and 8 of FIG. 10.

[0197] In embodiments, the composition comprises one of the formulations from the groups: 2, 3, 4, and 5 of FIG. 11.

[0198] In embodiments, the composition comprises one of the formulations of FIG. 13.

[0199] In embodiments, the composition comprises one of the formulations of FIG. 14.

[0200] In embodiments, the composition comprises one of the formulations from the groups: 2, 3, 4, 5, and 6 of FIG. 15.

[0201] In embodiments, the composition comprises one of the formulations of FIG. 19.

[0202] In embodiments, the composition has a weight to weight ratio of delivery lipid to one or more nucleic acidbased agents of about 5: 1 to about 15: 1 , or about 7: 1 to about 13: 1 , or about 8: 1 to about 12: 1 , or about 5: 1 , or about 6: 1 , or about 7: 1 , or about 8: 1 , or about 9: 1 , or about 10: 1 , or about 11 : 1 , or about 12: 1 , or about 13:1 , or about 14:1 , or about 15:1. In embodiments, the composition has a weight to weight ratio of delivery lipid to one or more nucleic acid-based agents from about 7:1 to about 13:1 . In embodiments, the composition has a weight to weight ratio of delivery lipid to one or more nucleic acid-based agents of about 7:1, or of about 13:1.

[0203] In embodiments, the composition has a weight to weight ratio of total lipid to one or more nucleic acid-based agents about 10:1 to about 35:1, about 15:1 to about 30:1 , about 20:1 to about 25:1 , or about 15:1 , or about 20:1 , or about 25:1, or about 30:1. In embodiments, the composition has a weight to weight ratio of total lipid to one or more nucleic acid-based agents from about 14:1 to about 26:1. In embodiments, the composition has a weight to weight ratio of total lipid to one or more nucleic acid-based agents of about 25:1 .

[0204] In embodiments, the composition forms a nucleic acid / lipid particle.

[0205] In embodiments, the one or more lipids associate into a lipid nanoparticle (LNP).

[0206] In embodiments, the LNP encapsulates the one or more nucleic acid-based agents.

[0207] Nucleic Acid-Based Agents: Mobile Element Enzyme

[0208] In embodiments, the one or more nucleic acid-based agents comprise a nucleic acid which encodes a mobile element enzyme. In embodiments, the mobile element enzyme has the amino acid sequence of SEQ ID NO: 1 , or an amino acid sequence having at least about 80%, or an amino acid sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

[0209] SEQ ID NO: 1 : Myotis lucifugus mobile element enzyme protein (572 amino acids, positions 2, 8, 13, and 125, shown in bold and underline)

[0210] MAQHSDYSDDEFCADKLSNYSCDSDLENASTSDEDSSDDEVMVRPRTLRRRRISSSSSDSESDIEGGREEW SHVDNPPVLEDFLGHQGLNTDAVINNIEDAVKLFIGDDFFEFLVEESNRYYNQNRNNFKLSKKSLKWKDIT PQEMKKFLGLIVLMGQVRKDRRDDYWTTEPWTETPYFGKTMTRDRFRQIWKAWHFNNNADIVNESDRLCKV RPVLDYFVPKFINIYKPHQQLSLDEGIVPWRGRLFFRVYNAGKIVKYGILVRLLCESDTGYICNMEIYCGE GKRLLETIQTWSPYTDSWYHIYMDNYYNSVANCEALMKNKFRICGTIRKNRGIPKDFQTISLKKGETKFI RKNDILLQVWQSKKPVYLISSIHSAEMEESQNIDRTSKKKIVKPNALIDYNKHMKGVDRADQYLSYYSILR RTVKWTKRLAMYMINCALFNSYAVYKSVRQRKMGFKMFLKQTAIHWLTDDIPEDMDIVPDLQPVPSTSGMR AKPPTSDPPCRLSMDMRKHTLQAIVGSGKKKNILRRCRVCSVHKLRSETRYMCKFCNIPLHKGACFEKYHT LKNY

[0211] In embodiments, the mobile element enzyme comprises an amino acid other than serine at the position corresponding to position 2 of SEQ ID NO: 1. In embodiments, the amino acid is a non-polar aliphatic amino acid, optionally a non-polar aliphatic amino acid optionally selected from G, A, V, L, I, and P, optionally A. In embodiments, the mobile element enzyme does not have additional residues at the C terminus relative to SEQ ID NO: 1. In embodiments, the enzyme has one or more mutations which confer hyperactivity. In embodiments, the enzyme has one or more amino acid substitutions selected from S8X1, C13X2, and N 125X3, or positions corresponding thereto relative to SEQ ID NO: 1 . In embodiments, the enzyme has S8X1 substitution, or at a position corresponding thereto relative to SEQ ID NO: 1. In embodiments, the enzyme has C13X2 substitution, or at a position corresponding thereto relative to SEQ ID NO: 1 . In embodiments, the enzyme has N125X3 substitution, or at a position corresponding thereto relative to SEQ ID NO: 1. In embodiments, Xi is selected from G, A, V, L, I, and P. In embodiments, X2 is selected from K, R, and H. In embodiments, X3 is selected K, R, and H. In embodiments, Xi is P and X2 is R.

[0212] In embodiments, the enzyme is capable of inserting a donor DNA at a TA dinucleotide site.

[0213] In embodiments, the enzyme is capable of inserting a donor DNA at a TTAA tetranucleotide site. In embodiments, the enzyme comprises: (a) a targeting element, and (b) an enzyme that is capable of inserting the donor DNA (e.g., a mobile element) comprising a gene, optionally at a TA dinucleotide site or a TTAA tetranucleotide site in a genomic safe harbor site (GSHS).

[0214] In embodiments, the targeting element targets the enzyme to a locus of interest. In embodiments, the targeting element comprises one or more of a gRNA, optionally associated with a Cas enzyme, which is optionally catalytically inactive or a transcription activator-like effector (TALE).

[0215] In embodiments, the targeting element comprises a transcription activator-like effector (TALE) DNA binding domain (DBD) and zinc fingers (ZF) DBD. In embodiments, the TALE DBD comprises one or more repeat sequences. In embodiments, the TALE DBD comprises about 14, or about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences. In embodiments, the TALE DBD repeat sequences comprise 33 or 34 amino acids. In embodiments, the TALE DBD repeat sequences comprise a repeat variable di-residue (RVD) at residue 12 or 13 of the 33 or 34 amino acids. In embodiments, the RVD recognizes one base pair in the nucleic acid molecule. In embodiments, the RVD recognizes a C residue in the nucleic acid molecule and is selected from HD, N(gap), HA, ND, and HI. In embodiments, the RVD recognizes a G residue in the nucleic acid molecule and is selected from NN, NH, NK, HN, and NA. In embodiments, the RVD recognizes an A residue in the nucleic acid molecule and is selected from Nl and NS. In embodiments, the RVD recognizes a T residue in the nucleic acid molecule and is selected from NG, HG, H(gap), and IG.

[0216] In embodiments, the targeting element comprises CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) associated protein 9 (Cas9), or a variant thereof. A CRISPR / Cas9 tool only requires Cas9 nuclease for DNA cleavage and a single-guide RNA (sgRNA) for target specificity. See Jinek et al. (2012) Science 337, 816-821; Chylinski et al. (2014) Nucleic Acids Res 42, 6091-6105. The inactivated form of Cas9, which is a nuclease-deficient (or inactive, or “catalytically dead” Cas9, is typically denoted as “dCas9,” has no substantial nuclease activity. Qi, L. S. et al. (2013). Cell 152, 1173-1183. CRISPR / dCas9 binds precisely to specific genomic sequences through targeting of guide RNA (gRNA) sequences. See Dominguez et al., Nat Rev Mol Cell Biol. 2016;17:5-15; Wang et al., Annu Rev Biochem. 2016;85:227-64. dCas9 is utilized to edit gene expression when applied to the transcription binding site of a desired site and / or locus in a genome. When the dCas9 protein is coupled to guide RNA (gRNA) to create dCas9 guide RNA complex, dCas9 prevents the proliferation of repeating codons and DNA sequences that might be harmful to an organism's genome. Essentially, when multiple repeat codons are produced, it elicits a response, or recruits an abundance of dCas9 to combat the overproduction of those codons and results in the shut-down of transcription. Thus, dCas9 works synergistically with gRNA and directly affects the DNA polymerase II from continuing transcription. In embodiments, the targeting element comprises a nuclease-deficient Cas enzyme guide RNA complex. In embodiments, the targeting element comprises a nuclease-deficient (or inactive, or “catalytically dead” Cas, e.g., Cas9, typically denoted as “dCas” or “dCas9”) guide RNA complex.

[0217] In embodiments, the targeting element comprises a Cas9 enzyme guide RNA complex. In embodiments, the Cas9 enzyme guide RNA complex comprises a nuclease-deficient dCas9 guide RNA complex.

[0218] Embodiments of the present disclosure make use of the ability of TALE or Cas or dCas9 / gRNA DBDs to target specific sites in a host genome. The DNA targeting ability of a TALE or Cas DBD or dCas9 / gRNA DBD is provided by TALE repeat sequences (e.g., modular arrays) or gRNA which are linked together to recognize flanking DNA sequences. Each TALE or gRNA can recognize certain base pair(s) or residue(s).

[0219] In embodiments, the GSHS is in an open chromatin location in a chromosome. In embodiments, the GSHS is selected from adeno-associated virus site 1 (AAVS1), chemokine (C-C motif) receptor 5 (CCR5) gene, HIV-1 coreceptor, and human Rosa26 locus.

[0220] In embodiments, the enzyme and the targeting element are connected. In embodiments, the enzyme and the targeting element are fused to one another or linked via a linker to one another. In embodiments, the linker is a flexible linker. In embodiments, the flexible linker is substantially comprised of glycine and serine residues, optionally wherein the flexible linker comprises (Gly4Ser)n, where n is from about 1 to about 12. In embodiments, the flexible linker is of about 20, or about 30, or about 40, or about 50, or about 60 amino acid residues.

[0221] Nucleic Acid-Based Agents: Donor DNA or Transgene or Mobile Element

[0222] In embodiments, the one or more nucleic acid-based agents comprise a donor DNA or transgene or mobile element.

[0223] In embodiments, the donor DNA or transgene or mobile element comprises a gene encoding a complete polypeptide. In embodiments, the donor DNA or transgene or mobile element comprises a gene which is defective or substantially absent in a disease state.

[0224] In embodiments, the donor DNA or transgene or mobile element is flanked by one or more ends. In embodiments, the mobile element enzyme recognizes the one or more ends.

[0225] In embodiments, the donor DNA or transgene or mobile element has a size of up to about 10 kb, or up to about 12 kb, up to about 15 kb, or about 7.5 to about 15 kb, or about 10 to about 15 kb. RNA / DNA and Ratios Thereof

[0226] In embodiments, the one or more nucleic acid-based agents is in the form of RNA. In embodiments, the RNA is or comprises mRNA. In embodiments, the mRNA is or comprises mmRNA. In embodiments, the mmRNA comprises one or more of a 5'-m7G cap (capO, cap1, or cap2), a pseudouridine or n-methyl-pseudouridine substitution, and a poly-A tail of about 30, or about 50, or about 100, of about 150 nucleotides in length.

[0227] In embodiments, the one or more nucleic acid-based agents is in the form of DNA. In embodiments, the DNA is or comprises plasmid DNA or miniplasmid DNA. In embodiments, the plasmid DNA has a size of up to about 10 kb, or up to about 12 kb, up to about 15 kb, or about 7.5 to about 15 kb, or about 10 to about 15 kb.

[0228] In embodiments, the nucleic acid-based agent comprises both a nucleic acid encoding a mobile element enzyme and a donor DNA or transgene or mobile element.

[0229] In embodiments, the nucleic acid-based agent comprises both RNA and DNA. In embodiments, the nucleic acid-based agent comprises both mRNA and DNA. In embodiments, the weight to weight ratio of DNA : mRNA is from about 10:1 to about 1 :10, or about 10 : about 1 , or about 9 : about 1 , or about 8 : about 1 , or about 7 : about 1 , or about 6 : about 1, or about 5 : about 1 , or about 4 : about 1 , or about 3 : about 1, or about 2 : about 1 , or about 1 : about 1 , or about 1 : about 2, or about 1 : about 3, or about 1 : about 4, or about 1 : about 5, or about 1 : about 6, or about 1 : about 7, or about 1 : about 8, or about 1 : about 9, or about 1 : about 10.

[0230] In embodiments, the nucleic acid-based agent comprises both mmRNA and plasmid DNA.

[0231] In embodiments, the nucleic acid-based agent comprises both a nucleic acid encoding a mobile element enzyme in the form of mmRNA and a donor DNA or transgene or mobile element in the form of plasmid DNA.

[0232] In embodiments, the nucleic acid-based agent comprises both a nucleic acid encoding a mobile element enzyme of SEQ ID NO: 1 in the form of mmRNA and a donor DNA or transgene or mobile element in the form of plasmid DNA.

[0233] In embodiments, the nucleic acid-based agent comprises both a nucleic acid encoding a mobile element enzyme in the form of mmRNA and a donor DNA or transgene or mobile element in the form of plasmid DNA and the weight to weight ratio of plasmid DNA : mmRNA is about 5 : about 1 , or about 2 : about 1 , or about 1 : about 1 , or about 1 : about 2, or about 1 : about 5. In embodiments, the nucleic acid-based agent comprises both a nucleic acid encoding a mobile element enzyme in the form of mmRNA and a donor DNA or transgene or mobile element in the form of plasmid DNA and the weight to weight ratio of plasmid DNA : mmRNA about 2 : about 1 .

[0234] Features of the LNP

[0235] In embodiments, the composition provides high encapsulation of the nucleic acid-based agent, e.g., compared a different collection of lipids, e.g., a different LNP.

[0236] In embodiments, the composition provides an encapsulation efficiency of greater than about 90%, greater than about 95%, greater than about 98%. Encapsulation efficiency can be measured as is known in the art, e.g., using fluorescent dye that is used in the detection and quantification of nucleic acids, e.g., using the RiboGreen assay (THERMO), or a similar assay.

[0237] In embodiments, the composition provides low polydispersity, e.g., compared a different collection of lipids, e.g., a different LNP. Assays / calculations used for the determination of polydispersity are known in the art, e.g., using parameters are defined in the ISO standard documents 13321:1996 E and ISO 22412:2008.

[0238] In embodiments, the composition is an LNP having a particle size of about 60 to about 200 nm, or about 100 to about 150 nm, or about 60 to about 120 nm. Assays / calculations used for the determination of particle size are known in the art, e.g., using parameters are defined in the ISO standard documents 13321 :1996 E and ISO 22412:2008.

[0239] Administration

[0240] In embodiments, the composition is suitable for systemic delivery in a subject. In embodiments, the composition is suitable for intravenous, subcutaneous, or intraperitoneal delivery in a subject.

[0241] In embodiments, the composition is suitable for local delivery in a subject. In embodiments, the composition is suitable for subretinal delivery in a subject.

[0242] Methods

[0243] In aspects, there is provided a method for introducing a nucleic acid into a cell, the method comprising contacting the cell with a composition described herein.

[0244] In aspects, there is provided a method for the in vivo delivery of a nucleic acid, the method comprising administering to a subject a composition described herein. In aspects, there is provided a method for ocular delivery of nucleic acid, the method comprising ocularly administering to a subject a composition described herein.

[0245] In aspects, there is provided a method for treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition described herein.

[0246] In aspects, there is provided a method for treating an ocular disease or disorder in a subject in need thereof, the method comprising administering to the subject’s retina a therapeutically effective amount of a composition described herein.

[0247] In embodiments, the method for treating an ocular disease or disorder in the subject in need thereof comprises administering to the subject’s retina a therapeutically effective amount of a composition comprising:

[0248] (a) one or more nucleic acid-based agents, and

[0249] (b) one or more lipids, the one or more lipids comprising:

[0250] (i) a delivery lipid,

[0251] (ii) cholesterol,

[0252] (iii) a helper lipid, and

[0253] (iv) a PEGylated lipid.

[0254] In embodiments, the one or more lipids comprises:

[0255] (i) a delivery lipid comprising about 15 mol %, about 16 mol%, about 17 mol%, about 18 mol%, about

[0256] 19 mol%, about 20 mol%, about 21 mol%, about 22 mol%, about 23 mol%, about 24 mol%, about

[0257] 25 mol%, about 26 mol%, about 27 mol%, about 28 mol%, about 29 mol%, about 30 mol%, about

[0258] 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about

[0259] 37 mol%, about 38 mol%, about 39 mol%, about 40 mol%, about 41 mol%, about 42 mol%, about

[0260] 43 mol%, about 44 mol%, or about 45 mol % of the total lipid present, the delivery lipid being SS- OP,

[0261] (ii) a cholesterol comprising about 35 mol %, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, 45 mol %, about 46 mol%, about 47 mol%, about 48 mol%, about 49 mol%, about 50 mol%, about 51 mol%, about 52 mol%, about 53 mol%, about 54 mol%, about 55 mol %, about 56 mol%, about 57 mol%, about 58 mol%, about 59 mol%, or about 60 mol % of the total lipid present,

[0262] (iii) a helper lipid comprising about 5 mol %, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, about 15 mol %, about 16 mol%, about 17 mol%, about 18 mol%, about 19 mol%, about 20 mol%, about 21 mol%, about 22 mol%, about 23 mol%, about 24 mol%, about 25 mol%, about 26 mol%, about 27 mol%, about 28 mol%, about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, or about 35 mol % of the total lipid present, optionally being one or more of DOPE or DSPC, and

[0263] (iv) a PEGylated lipid comprising from about 3 mol %, about 4 mol%, about 5 mol%, about 6 mol%, or about 7 mol % of the total lipid present, optionally being PEG-2K-DMG.

[0264] In embodiments, the one or more lipids comprises:

[0265] (i) a delivery lipid comprising from about 22 mol % to about 26 mol % of the total lipid present, the delivery lipid being SS-OP,

[0266] (ii) cholesterol comprising from about 46 mol % to about 50 mol % of the total lipid present,

[0267] (iii) a helper lipid comprising from about 22 mol % to about 26 mol % of the total lipid present, optionally being one or more of DOPE or DSPC, and

[0268] (iv) a PEGylated lipid comprising from about 4 mol % to about 6 mol % of the total lipid present, optionally being PEG-2K-DMG.

[0269] In embodiments, the one or more lipids comprises:

[0270] (i) a delivery lipid comprising from about 34 mol % to about 38 mol % of the total lipid present, the delivery lipid being SS-OP,

[0271] (ii) cholesterol comprising from about 48 mol % to about 52 mol % of the total lipid present,

[0272] (iii) a helper lipid comprising from about 8 mol % to about 10 mol % of the total lipid present, optionally being one or more of DOPE or DSPC, and

[0273] (iv) a PEGylated lipid comprising from about 4 mol % to about 6 mol % of the total lipid present, optionally being PEG-2K-DMG. In embodiments, the one or more lipids comprises:

[0274] (i) a delivery lipid comprising about 36 mol % of the total lipid present, the delivery lipid being SS- OP,

[0275] (ii) cholesterol comprising about 51 mol % of the total lipid present,

[0276] (iii) a helper lipid comprising about 10 mol % of the total lipid present, optionally being one or more of DOPE or DSPC, and

[0277] (iv) a PEGylated lipid comprising about 5 mol % of the total lipid present, optionally being PEG-2K- DMG.

[0278] In embodiments, the one or more lipids comprises:

[0279] (i) a delivery lipid comprising about 36 mol % of the total lipid present, the delivery lipid being SS- OP,

[0280] (ii) cholesterol comprising about 51 mol % of the total lipid present,

[0281] (iii) a helper lipid comprising about 10 mol % of the total lipid present, optionally being one or more of DOPE or DSPC, and

[0282] (iv) a PEGylated lipid comprising about 5 mol % of the total lipid present, optionally being PEG-2K- DMG, and a nucleic acid-based agent having a weight to weight ratio of delivery lipid to one or more nucleic acid-based agents of about 7:1 and / or a having a weight to weight ratio of total lipid to one or more nucleic acid-based agents of about 25:1 .

[0283] In embodiments, the one or more lipids comprises:

[0284] (i) a delivery lipid comprising about 24 mol % of the total lipid present, the delivery lipid being SS- OP,

[0285] (ii) cholesterol comprising about 48 mol % of the total lipid present,

[0286] (iii) a helper lipid comprising about 24 mol % of the total lipid present, optionally being one or more of DOPE or DSPC, and

[0287] (iv) a PEGylated lipid comprising about 5 mol % of the total lipid present, optionally being PEG-2K- DMG. In embodiments, the one or more lipids comprises:

[0288] (i) a delivery lipid comprising about 24 mol % of the total lipid present, the delivery lipid being SS- OP,

[0289] (ii) cholesterol comprising about 48 mol % of the total lipid present,

[0290] (iii) a helper lipid comprising about 24 mol % of the total lipid present, optionally being one or more of DOPE or DSPC, and

[0291] (iv) a PEGylated lipid comprising about 5 mol % of the total lipid present, optionally being PEG-2K- DMG, and a nucleic acid-based agent having a weight to weight ratio of delivery lipid to one or more nucleic acid-based agents of about 7:1 and / or a having a weight to weight ratio of total lipid to one or more nucleic acid-based agents of about 25:1 .

[0292] In embodiments, the one or more lipids comprise a dual nucleic acid-based agent as a cargo. In embodiments, the one or more lipids comprising a dual nucleic acid-based agent comprise SS-OP as a delivery lipid.

[0293] In embodiments, the one or more lipids comprises:

[0294] (i) a delivery lipid comprising about 15 mol %, about 16 mol%, about 17 mol%, about 18 mol%, about 19 mol%, about 20 mol%, about 21 mol%, about 22 mol%, about 23 mol%, about 24 mol%, about 25 mol%, about 26 mol%, about 27 mol%, about 28 mol%, about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, or about 35 mol% of the total lipid present, the delivery lipid being SS-EC,

[0295] (ii) cholesterol comprising about 35 mol %, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, 45 mol %, about 46 mol%, about 47 mol%, about 48 mol%, about 49 mol%, about 50 mol%, about 51 mol%, about 52 mol%, about 53 mol%, about 54 mol%, about 55 mol %, about 56 mol%, about 57 mol%, about 58 mol%, about 59 mol%, or about 60 mol % of the total lipid present,

[0296] (iii) a helper lipid comprising about 15 mol %, about 16 mol%, about 17 mol%, about 18 mol%, about 19 mol%, about 20 mol%, about 21 mol%, about 22 mol%, about 23 mol%, about 24 mol%, about 25 mol%, about 26 mol%, about 27 mol%, about 28 mol%, about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, or about 35 mol % of the total lipid present, optionally being one or more of DOPE or DSPC, and

[0297] (iv) a PEGylated lipid comprising from about 3 mol %, about 4 mol%, about 5 mol%, about 6 mol%, or about 7 mol % of the total lipid present, optionally being PEG-2K-DMG.

[0298] In embodiments, the one or more lipids comprises:

[0299] (i) a delivery lipid comprising from about 22 mol % to about 26 mol % of the total lipid present, the delivery lipid being SS-EC,

[0300] (ii) cholesterol comprising from about 46 mol % to about 50 mol % of the total lipid present,

[0301] (iii) a helper lipid comprising from about 22 mol % to about 26 mol % of the total lipid present, optionally being one or more of DOPE or DSPC, and

[0302] (iv) a PEGylated lipid comprising from about 4.5 mol % to about 5.5 mol % of the total lipid present, optionally being PEG-2K-DMG.

[0303] In embodiments, the one or more lipids comprises:

[0304] (i) a delivery lipid comprising about 24 mol % of the total lipid present, the delivery lipid being SS- EC,

[0305] (ii) cholesterol comprising about 48 mol % of the total lipid present,

[0306] (iii) a helper lipid comprising about 24 mol % of the total lipid present, optionally being one or more of DOPE or DSPC, and

[0307] (iv) a PEGylated lipid comprising about 5 mol % of the total lipid present, optionally being PEG-2K- DMG.

[0308] In embodiments, the one or more lipids comprises:

[0309] (i) a delivery lipid comprising about 24 mol % of the total lipid present, the delivery lipid being SS- EC,

[0310] (ii) cholesterol comprising about 48 mol % of the total lipid present,

[0311] (iii) a helper lipid comprising about 24 mol % of the total lipid present, optionally being one or more of DOPE or DSPC, and (iv) a PEGylated lipid comprising about 5 mol % of the total lipid present, optionally being PEG-2K- DMG, and a nucleic acid-based agent having a weight to weight ratio of delivery lipid to one or more nucleic acid-based agents of about 7:1 and / or a having a weight to weight ratio of total lipid to one or more nucleic acid-based agents of about 25:1 .

[0312] Kits

[0313] In embodiments, the compositions, or one or more lipids, optionally with the nucleic acid-based agents, is included in a container, kit, pack, or dispenser together with instructions for administration and / or association (e.g., to form an LNP).

[0314] In embodiments, the kit includes instructions for administration and / or association (e.g., to form an LNP) in accordance with the method of the present disclosure.

[0315] Definitions

[0316] The following definitions are used in connection with the disclosure disclosed herein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of skill in the art to which this invention belongs.

[0317] As used herein, “a,” “an,” or “the” can mean one or more than one.

[0318] Further, the term “about” when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 10% of that referenced numeric indication. For example, the language “about 50” covers the range of 45 to 55.

[0319] An “effective amount,” when used in connection with medical uses is an amount that is effective for providing a measurable treatment, prevention, or reduction in the rate of pathogenesis of a disease of interest.

[0320] As used herein, the term “variant” encompasses but is not limited to nucleic acids or proteins which comprise a nucleic acid or amino acid sequence which differs from the nucleic acid or amino acid sequence of a reference by way of one or more substitutions, deletions and / or additions at certain positions. The variant may comprise one or more conservative substitutions. Conservative substitutions may involve, e.g., the substitution of similarly charged or uncharged amino acids.

[0321] As referred to herein, all compositional percentages are by weight of the total composition, unless otherwise specified. As used herein, the word “include,” and its variants, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the compositions and methods of this technology. Similarly, the terms “can” and “may” and their variants are intended to be non-limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features.

[0322] Although the open-ended term “comprising,” as a synonym of terms such as including, containing, or having, is used herein to describe and claim the invention, the present invention, or embodiments thereof, may alternatively be described using alternative terms such as “consisting of or “consisting essentially of.”

[0323] As used herein, “methods of treatment” are equally applicable to use of a composition for treating the diseases or disorders described herein and / or compositions for use and / or uses in the manufacture of a medicaments for treating the diseases or disorders described herein.

[0324] SELECTED SEQUENCES

[0325] In embodiments, the present disclosure provides for any of the sequence provided herein, including the below, and a variant sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.

[0326] SEQ ID NO: 1 : Myotis lucifugus mobile element enzyme protein (572 amino acids, positions 2, 8, 13, and 125, described in the text, reshown in bold and underline)

[0327] MAQHSDYSDDEFCADKLSNYSCDSDLENASTSDEDSSDDEVMVRPRTLRRRRISSSSSDSESDIEGGREEW SHVDNPPVLEDFLGHQGLNTDAVINNIEDAVKLFIGDDFFEFLVEESNRYYNQNRNNFKLSKKSLKWKDIT PQEMKKFLGLIVLMGQVRKDRRDDYWTTEPWTETPYFGKTMTRDRFRQIWKAWHFNNNADIVNESDRLCKV RPVLDYFVPKFINIYKPHQQLSLDEGIVPWRGRLFFRVYNAGKIVKYGILVRLLCESDTGYICNMEIYCGE GKRLLETIQTWSPYTDSWYHIYMDNYYNSVANCEALMKNKFRICGTIRKNRGIPKDFQTISLKKGETKFI RKNDILLQVWQSKKPVYLISSIHSAEMEESQNIDRTSKKKIVKPNALIDYNKHMKGVDRADQYLSYYSILR RTVKWTKRLAMYMINCALFNSYAVYKSVRQRKMGFKMFLKQTAIHWLTDDIPEDMDIVPDLQPVPSTSGMR AKPPTSDPPCRLSMDMRKHTLQAIVGSGKKKNILRRCRVCSVHKLRSETRYMCKFCNIPLHKGACFEKYHT LKNY

[0328] Constructs for ABCA4 nucleic acids delivery

[0329] ATP binding cassette subfamily A member 4 (ABCA4) protein is a retina-specific ABC transporter with N- retinylidene-PE as a substrate. It is expressed exclusively in retina photoreceptor cells, and the gene product mediates transport of an essential molecule, all-trans-reti nal aldehyde (atRAL), across the photoreceptor cell membrane. Mutations in this gene are found in patients diagnosed with Stargardt disease, a form of juvenileonset macular degeneration. Mutations in this gene are also associated with other conditions such as retinitis pigmentosa-19, cone-rod dystrophy type 3, early-onset severe retinal dystrophy, fundus flavimaculatus, and macular degeneration age-related 2.

[0330] In embodiments, the ABCA4 nucleic acid is operably coupled to a promoter that can influence overall expression levels and cell-specificity of the transgenes (e.g. ABCA4 or a functional fragment thereof).

[0331] In some embodiments, the promoter is a CAG promoter (cytomegalovirus (CMV) enhancer fused to the chicken p-actin promoter and rabbit beta-Globin splice acceptor) (1732 bp), which expresses in both RPE and photoreceptor levels in vivo and in vitro. In some embodiments, the CAG promoter comprises the following nucleotide sequence.

[0332] Nucleic acid-based agent

[0333] (1) SG-D2-0034 Cag-hABCA4 (lnsulator.Cag.hABCA4-CO.rBG. Insulator)

[0334] Left Mobile element enzyme ITR (SEQ ID NO: 2)

[0335] TTAACACTTGGATTGCGGGAAACGAGTTAAGTCGGCTCGCGTGAATTGCGCGTACTCCGCGGGAGCC GTCTTAACTCGGTTCATATAGATTTGCGGTGGAGTGCGGGAAACGTGTAAACTCGGGCCGATTGTAAC TGCGTATTACCAAATATTTGTT

[0336] Spacer sequence 1 (SEQ ID NO: 3)

[0337] AGATCCTCTAGCGATAAGCTTGATATCGAATTCGAGGTCGACT

[0338] Dimer HS4 Insulator (SEQ ID NO: 4)

[0339] GGCGCGCCTGTCATTCTAAATCTCTCTTTCAGCCTAAAGCTTTTTCCCCGTATCCCCCCAGGTGTCTGC AGGCTCAAAGAGCAGCGAGAAGCGTTCAGAGGAAAGCGATCCCGTGCCACCTTCCCCGTGCCCGGG CTGTCCCCGCACGCTGCCGGCTCGGGGATGCGGGGGGAGCGCCGGACCGGAGCGGAGCCCCGGG CGGCTCGCTGCTGCCCCCTAGCGGGGGAGGGACGTAATTACATCCCTGGGGGCTTTGGGAGGGGGC TGTCCCCGTGAGCTCCCAGGCGCGCCTGTCATTCTAAATCTCTCTTTCAGCCTAAAGCTTTTTCCCCGT ATCCCCCCAGGTGTCTGCAGGCTCAAAGAGCAGCGAGAAGCGTTCAGAGGAAAGCGATCCCGTGCCA

[0340] CCTTCCCCGTGCCCGGGCTGTCCCCGCACGCTGCCGGCTCGGGGATGCGGGGGGAGCGCCGGACC GGAGCGGAGCCCCGGGCGGCTCGCTGCTGCCCCCTAGCGGGGGAGGGACGTAATTACATCCCTGG GGGCTTTGGGGGGGGGCTGTCCCCGT

[0341] Spacer Sequence 2 (SEQ ID NO: 5)

[0342] TCTAGAGAGCTCAATAAAAGAGCCCACAACCCCTCACTCGGC Cag Promoter (combination of the CMV Enhancer, Chicken B-Actin Promoter, and Chimeric Intron)

[0343] CMV Enhancer (SEQ ID NO: 6)

[0344] GTCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATAT

[0345] GGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCA

[0346] TTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTG

[0347] GAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATT

[0348] GACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTAC

[0349] TTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATG

[0350] Chicken B-Actin Promoter (SEQ ID NO: 7)

[0351] GTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTA

[0352] TTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGC

[0353] GGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAG

[0354] AGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGA

[0355] AGCGCGCGGCGGGCG

[0356] Chimeric Intron (SEQ ID NO: 8)

[0357] GGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCG GCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAAT TAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGA GGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCG CGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTC CGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAG GGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGT CGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGG GCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTG CCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGG AGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGG GCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCT CTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCG TGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTG CCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCT CTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGT CTCATCATTTTGGCAAAGAATTC attB1 / Partial Kozak Sequence (SEQ ID NO: 9)

[0358] CAAGTTTGTACAAAAAAGCAGGCTGCCACC ABCA4 (Hs) Codon Optimized NM_000350.3 (SEQ ID NO: 10)

[0359] ATGGGTTTCGTGCGCCAGATCCAGCTGCTGCTTTGGAAGAACTGGACCCTGAGAAAAAGGCAGAAGA TCAGGTTCGTGGTGGAGCTGGTGTGGCCTCTGAGTCTGTTTCTGGTGCTGATCTGGCTGAGGAACGC CAACCCACTTTACAGTCACCACGAATGCCATTTTCCCAATAAGGCCATGCCAAGCGCCGGCATGCTGC CATGGCTGCAGGGCATCTTCTGCAATGTGAACAACCCTTGCTTCCAGAGCCCAACCCCCGGCGAGAG CCCTGGCATCGTGTCTAATTATAACAACTCCATCCTGGCCAGGGTGTACCGTGATTTTCAGGAACTGC

[0360] TGATGAATGCCCCCGAGAGCCAGCATCTGGGCAGGATCTGGACAGAGCTCCATATTCTGTCACAGTTT ATGGATACTTTGCGCACTCACCCTGAACGCATCGCCGGCAGAGGCATCAGGATCCGCGACATCTTGA AGGACGAAGAAACCCTGACCCTGTTTCTCATCAAAAATATCGGCCTGTCCGATTCCGTGGTGTATCTTC TGATTAATAGCCAGGTGAGGCCCGAACAGTTCGCCCACGGCGTGCCTGATCTGGCTCTGAAGGACAT TGCCTGTAGCGAGGCCCTGCTGGAGCGCTTTATCATTTTCTCCCAGCGGAGGGGCGCCAAAACTGTG

[0361] CGCTACGCCCTGTGCTCTCTGAGCCAGGGCACCCTGCAGTGGATCGAGGATACTCTGTATGCCAACG TCGACTTCTTTAAGCTGTTCCGGGTGCTGCCAACCCTCCTGGACAGTCGGAGCCAGGGAATCAATCTG CGGTCCTGGGGCGGCATTCTGTCCGACATGTCACCTAGAATACAGGAGTTCATCCACAGGCCATCCAT GCAGGACCTGCTGTGGGTGACAAGGCCACTGATGCAGAACGGCGGCCCTGAGACATTCACAAAGCTG ATGGGAATCTTGTCTGACCTGCTGTGTGGGTATCCAGAGGGGGGGGGCTCCCGGGTGCTGAGCTTTA

[0362] ACTGGTACGAGGACAATAATTACAAAGCCTTTCTCGGCATCGACTCTACCAGAAAGGACCCGATTTATT CTTACGACAGGAGGACCACCAGCTTCTGCAATGCCCTGATCCAGAGCCTGGAGTCCAACCCTCTGAC AAAAATTGCCTGGAGAGCAGCCAAGCCTCTGCTGATGGGAAAAATCCTGTACACCCCCGATTCACCTG CCGCCAGGCGCATCCTCAAGAACGCAAATAGTACCTTCGAGGAACTCGAGCACGTGAGGAAACTGGT GAAGGCCTGGGAGGAGGTGGGACCTCAGATCTGGTACTTTTTCGATAATTCTACACAGATGAATATGA

[0363] TCAGAGATACACTGGGCAATCCTACCGTGAAGGACTTCCTGAATAGACAGCTGGGCGAGGAGGGCAT CACTGCCGAAGCCATTCTGAACTTCCTGTACAAGGGACCAAGGGAGAGCCAAGCAGATGACATGGCC AACTTCGATTGGAGAGATATTTTCAACATCACTGATCGGACCCTGAGGCTGGTGAATCAGTACCTGGA ATGCCTGGTGCTGGACAAGTTCGAGTCCTACAATGACGAGACCCAGCTGACACAGCGGGCACTGAGC CTGCTCGAGGAAAATATGTTTTGGGCAGGGGTGGTGTTCCCCGACATGTACCCATGGACTAGCTCTCT

[0364] GCCCCCCCACGTGAAATATAAGATCAGAATGGACATCGACGTGGTGGAGAAGACCAACAAGATCAAA GACAGATACTGGGATAGTGGGCCACGCGCAGACCCCGTGGAGGATTTCCGCTACATCTGGGGCGGC TTCGCCTACCTGCAGGACATGGTGGAACAGGGCATCACTCGCAGCCAGGTGCAGGCCGAGGCACCC

[0365] GTGGGCATCTACCTGCAGCAGATGCCCTACCCCTGCTTCGTGGACGATTCATTCATGATCATTCTGAA CCGCTGTTTTCCCATTTTTATGGTGTTGGCCTGGATCTACAGCGTGTCCATGACCGTGAAATCCATCGT GCTGGAGAAGGAGCTCAGGCTGAAGGAGACCCTGAAGAACCAGGGTGTGTCAAATGCCGTGATCTG

[0366] GTGCACCTGGTTCCTGGATTCCTTTTCCATCATGAGCATGTCTATCTTCCTGCTGACCATCTTTATTATG CATGGGAGGATTCTCCATTACTCCGATCCCTTTATCCTGTTCCTGTTCCTGCTGGCCTTCTCTACCGCC ACCATCATGCTGTGCTTCCTGCTGAGCACCTTTTTTAGCAAGGCCTCCCTGGCCGCCGCCTGCAGCG GCGTGATTTACTTTACCCTGTACCTGCCCCATATTCTGTGTTTTGCATGGCAGGACAGAATGACCGCC

[0367] GAGCTGAAGAAGGCCGTGAGTCTGCTGTCTCCTGTGGCCTTCGGGTTTGGGACCGAGTACCTGGTGC GGTTCGAGGAGCAGGGCCTCGGCCTCCAGTGGAGCAACATCGGCAACAGCCCCACAGAGGGAGACG AGTTCTCTTTCCTGCTGTCCATGCAGATGATGCTGCTGGACGCCGCTGTGTACGGCCTGCTCGCCTG

[0368] GTATCTGGACCAGGTGTTCCCCGGGGATTATGGCACTCCTCTGCCCTGGTACTTCCTGCTGCAAGAAA GCTACTGGCTGGGCGGGGAGGGGTGTTCCACTCGCGAGGAGCGCGCCCTGGAAAAGACAGAACCCC TGACAGAGGAGACCGAAGATCCCGAGCATCCTGAGGGCATACACGACTCATTCTTCGAGCGCGAGCA CCCAGGCTGGGTGCCAGGCGTGTGTGTGAAAAATCTTGTCAAAATTTTCGAACCTTGCGGTAGACCTG CAGTGGACAGACTCAACATAACATTCTACGAGAACCAGATCACCGCCTTCCTGGGACACAACGGCGC CGGCAAAACCACCACCCTGAGCATCCTGACAGGCCTGCTCCCTCCAACCAGTGGAACCGTGCTGGTT

[0369] GGAGGCAGAGACATCGAGACCAGCCTGGACGCCGTCCGGCAGAGCCTGGGCATGTGCCCTCAGCAC

[0370] AACATTCTCTTCCACCACCTGACCGTGGCCGAGCACATGCTCTTCTACGCCCAGCTGAAGGGCAAGA

[0371] GCCAGGAAGAGGCCCAGCTCGAGATGGAGGCTATGCTGGAGGACACAGGCCTGCACCATAAGAGAA

[0372] ACGAAGAGGCACAGGATCTGAGCGGCGGCATGCAGAGGAAACTGTCCGTGGCCATCGCCTTCGTGG

[0373] GGGACGCTAAAGTTGTGATCCTGGACGAGCCCACATCAGGGGTGGACCCCTATAGCAGGCGGTCCAT

[0374] CTGGGACCTTCTGCTGAAGTATCGCAGTGGACGCACAATCATCATGTCAACCCATCACATGGACGAGG

[0375] CTGACCTGCTGGGCGACCGCATCGCTATCATCGCCCAGGGCAGGCTGTATTGCTCTGGGACTCCTCT

[0376] GTTTCTGAAGAACTGCTTTGGCACCGGCCTGTACCTGACACTGGTGCGGAAAATGAAAAATATACAGA

[0377] GCCAGAGAAAGGGCAGCGAGGGAACCTGCAGCTGTTCTTCCAAGGGCTTTTCCACCACCTGTCCTGC

[0378] GCACGTGGACGACCTGACCCCCGAGCAGGTGCTGGACGGCGACGTGAACGAGCTGATGGACGTGGT

[0379] GCTGCACCATGTGCCCGAGGCCAAGCTGGTGGAGTGTATCGGGCAGGAGCTGATCTTTCTGTTGCCA

[0380] AATAAGAATTTCAAACATAGGGCCTACGCATCCCTGTTCCGGGAGCTCGAAGAGACCCTGGCTGATCT

[0381] GGGACTGTCCTCTTTCGGGATCTCCGATACCCCCCTGGAGGAGATTTTTCTGAAAGTGACCGAGGATA

[0382] GCGATTCTGGGCCACTGTTTGCCGGGGGCGCTCAGCAGAAGAGGGAGAATGTGAACCCCAGACACC

[0383] CTTGCCTGGGCCCCAGGGAGAAGGCTGGCCAGACACCTCAGGACTCTAACGTGTGCTCCCCTGGCG

[0384] CCCCCGCCGCCCATCCCGAGGGCCAACCCCCTCCCGAGCCCGAATGCCCTGGCCCCCAGCTGAATA

[0385] CCGGAACTCAGCTGGTGCTTCAGCATGTGCAGGCACTGCTGGTCAAACGGTTCCAGCACACCATCAG

[0386] GTCCCACAAAGACTTCCTGGCCCAGATCGTGCTGCCAGCCACATTTGTGTTCCTGGCCCTGATGCTGT

[0387] CTATCGTGATACCTCCTTTTGGCGAGTACCCCGCTCTCACCCTGCACCCTTGGATTTACGGCCAGCAG

[0388] TATACATTCTTCTCCATGGACGAGCCAGGAAGCGAGCAGTTCACCGTGCTGGCCGACGTGCTGCTGA

[0389] ACAAGCCAGGGTTCGGCAACAGGTGCCTGAAAGAAGGATGGCTGCCTGAGTATCCCTGCGGAAACAG

[0390] CACACCCTGGAAGACTCCAAGCGTGTCTCCCAACATCACACAGCTGTTCCAGAAGCAGAAGTGGACTC

[0391] AGGTGAATCCCTCCCCCAGCTGTAGATGTTCCACCAGAGAGAAACTGACGATGCTGCCAGAGTGTCCT

[0392] GAGGGAGCCGGCGGGCTGCCCCCTCCTCAGCGCACTCAGCGGAGCACCGAAATCCTGCAGGACCTG

[0393] ACAGATCGGAACATCTCTGACTTTCTGGTTAAGACCTACCCCGCCCTGATCAGGTCTAGCCTGAAGAG

[0394] CAAATTCTGGGTGAATGAGCAGCGCTATGGAGGCATCTCCATTGGAGGCAAGCTGCCAGTGGTGCCC

[0395] ATTACAGGTGAGGCCCTCGTGGGCTTCCTGTCCGACCTGGGCAGAATTATGAACGTGTCAGGAGGAC

[0396] CTATCACCCGCGAGGCCTCCAAGGAGATCCCTGATTTCCTCAAGCACCTGGAGACCGAAGACAACATT

[0397] AAGGTGTGGTTCAATAACAAGGGCTGGCACGCCCTGGTGTCCTTCCTGAATGTGGCTCACAACGCCAT

[0398] CCTGCGGGCCTCTCTGCCAAAGGACAGGTCTCCTGAGGAGTACGGAATAACAGTGATCTCTCAGCCA

[0399] CTGAACCTCACCAAGGAGCAGCTGAGCGAAATTACTGTGCTGACCACCAGCGTGGACGCCGTGGTGG

[0400] CCATCTGTGTAATCTTTTCCATGTCATTCGTGCCCGCTTCCTTCGTGCTCTACCTGATCCAGGAGAGGG

[0401] TGAATAAGTCTAAGCATTTGCAGTTCATCTCTGGTGTCTCCCCCACTACCTACTGGGTGACGAATTTCC

[0402] TGTGGGACATCATGAACTACTCCGTGAGTGCCGGCCTGGTGGTGGGCATTTTCATCGGATTCCAGAA

[0403] GAAGGCCTATACCAGCCCTGAGAATCTGCCTGCCCTTGTGGCACTGCTGCTCCTGTACGGATGGGCC

[0404] GTGATCCCTATGATGTACCCTGCCTCCTTCCTGTTCGATGTGCCCAGCACCGCCTACGTGGCCCTGTC

[0405] ATGCGCCAACCTGTTCATTGGGATCAATAGCTCCGCCATCACCTTTATCCTGGAGCTGTTCGAGAACA

[0406] ACCGCACCCTGCTGCGCTTCAACGCCGTGCTGAGGAAATTGCTGATCGTGTTTCCCCACTTTTGCCTG

[0407] GGCCGGGGCCTGATTGACCTGGCCCTGTCACAGGCCGTGACCGACGTGTATGCCCGGTTCGGCGAG

[0408] GAGCATTCTGCCAACCCCTTCCACTGGGACCTGATCGGCAAGAACTTGTTCGCCATGGTGGTGGAGG

[0409] GCGTGGTGTATTTCCTGCTGACTTTGCTGGTCCAGAGGCATTTCTTCCTGAGCCAGTGGATCGCCGAG

[0410] CCAACAAAGGAACCCATCGTGGACGAGGACGACGACGTCGCCGAAGAGCGGCAGAGGATCATCACC

[0411] GGCGGCAACAAGACAGACATCCTGAGACTGCATGAGCTCACCAAGATCTATCCCGGAACATCCAGCC

[0412] CTGCCGTGGATAGGTTGTGTGTGGGCGTGCGGCCAGGCGAGTGTTTCGGACTGCTGGGCGTGAATG

[0413] GTGCCGGCAAAACAACAACCTTTAAGATGCTGACTGGCGACACCACCGTGACCAGTGGCGATGCGAC

[0414] CGTGGCCGGCAAGTCCATCCTGACTAACATTTCAGAGGTGCACCAGAACATGGGGTATTGCCCCCAG TTTGACGCCATTGATGAGCTGCTGACTGGCAGGGAGCACCTCTACCTGTATGCGCGTCTGCGGGGCG

[0415] TGCCTGCCGAGGAAATCGAAAAGGTGGCCAACTGGTCTATTAAATCACTGGGCCTGACCGTGTACGC

[0416] AGATTGCCTGGCCGGCACCTACTCAGGCGGTAACAAGAGGAAGCTGTCAACAGCAATCGCCCTGATC

[0417] GGCTGCCCTCCTTTGGTGCTGCTGGATGAACCCACAACCGGAATGGACCCCCAGGCCCGGAGAATGC

[0418] TGTGGAACGTGATCGTGTCCATCATCAGGGAAGGCCGGGCCGTGGTGCTGACCAGCCACAGCATGG

[0419] AAGAGTGCGAAGCCCTGTGCACACGCCTGGCAATCATGGTGAAGGGCGCCTTCCGATGTATGGGCAC

[0420] CATCCAGCACCTGAAGAGCAAGTTCGGTGACGGCTATATCGTGACTATGAAGATCAAGAGCCCCAAAG

[0421] ATGATCTGCTGCCTGACCTGAACCCAGTGGAGCAGTTCTTCCAGGGCAATTTCCCAGGCAGCGTGCA

[0422] GCGAGAGAGACACTACAACATGCTGCAGTTCCAGGTGTCCTCCTCATCCCTGGCACGGATCTTTCAGC

[0423] TGCTGCTGTCCCACAAGGACAGCCTGCTGATCGAAGAGTACTCTGTGACACAGACTACACTGGACCA

[0424] GGTGTTCGTGAATTTCGCTAAGCAGCAGACCGAAAGTCACGACCTGCCTCTGCATCCACGGGCCGCC

[0425] GGGGCCAGTAGACAGGCTCAGGATTGA attB2 (SEQ ID NO: 11)

[0426] ACCCAGCTTTCTTGTACAAAGTGG rBG PA (includes B-globin Poly(A) Signal) (SEQ ID NO: 12)

[0427] TCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATAC

[0428] CACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCT

[0429] GGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGA

[0430] CATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCC

[0431] ATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCT

[0432] GTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATT

[0433] TTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTAC

[0434] TCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATC

[0435] D4Z4-C insulator (SEQ ID NO: 13)

[0436] CCTCCTGGCTGCACCTGCCGCAGTGCACAGTCCGGCTGAGGTGCACGGGAGCCCGCCGGCCTCTCT

[0437] CTGCCCGCGTCCGTCCGTGAAATTCCGGCCGGGGCTCACCGCGATGGCCCTCCCGACACCCTCGGA

[0438] CAGCACCCTCCCCGCGGAAGCCCGGGGACGAGGACGGCCACGGAGACTCGTTTGGACCCCGAGCC

[0439] AAAGCGAGGCCCTGCGAGCCTGCTTTGAGCGGAACCCGTACCCGGGCATCGCCACCAGAGAACGGC

[0440] TGGCCCAGGCCATCGGCATTCCGGAGCCCAGGGTCCAGATTTGGTTTCAGAATGAGAGGTCACGCCA

[0441] GCTGAGGCAGCACCGGCGGGAATCTCGGCCCTGGCCCGGGAGACGCGGCCCGCCAGAAGGCCGGC

[0442] GAAAGCGGACCGCCGTCACCGGATCCCAGACCGCCCTGCTCCTCCGAGCCTTTGAGAAGGATCGCTT

[0443] TCCAGGCATCGCCGCCCGGGAGGAGCTGGCCAGAGAGACGGGCCTCCCGGAGTCCAGGATTCAGAT

[0444] CTGGTTTCAGAATCGAAGGGCCAGGCACCCGGGACAGGGTGGCAGGGCGCCCGCGCAGGCAGGCG

[0445] GCCTGTGCAGCGCGGCCCCCGGCGGGGGTCACCCTGCTCCCTCGTGGGTCGCCTTCGCCCACACCG

[0446] GCGCGTGGGGAACGGGGCTTCCCGCACCCCACGTGCCCTGCGCGCCTGGGGCTCTCCCACAGGGG

[0447] GCTTTCGTGAGCCAGGCAGCGAGGGCCGCCCCCGCGCTGCAGCCCAGCCAGGCCGCGCCGGCAGA

[0448] GGGGGTCTCCCAACCTGCCCCGGCGCGCGGGGATTTCGCCTACGCCGCCCCGGCTCCTCCGGAGC CGGGGCGCTCTCCCACCCTCAGGCTCCTCGGTGGCCTCCGCACCCGGGCAAAAGCCGGGAGGACC

[0449] GGGACCCGCAGCGCGACGGCCTGCCGGGCCCCTGCGCGGTGGCACAGCCTGGGCCCGCTCAAGCG

[0450] GGGCCGCAGGCCAAGGGGTGCTTGCGCCACCCACGTCCCAGGGGAGTCCGTGGTGGGGCTGGGGC

[0451] CGGGGTCCCCAGGTCGCCGGGGCGGCGTGGGAACCCCAAGCCGGGGCAGCTCCACCTCCCCAGCC

[0452] CGCGCCCCCGGGACGCCTCCGCCTCCGCGCGGCAGGGGCAGATGCAAGGCATCCCGGCGCCCTCC

[0453] CAGGCGCTCCAGGAGCCGGCGCCCTGGTCTGCACTCCCCTGCGGCCTGCTGCTGGATGAGCTCCTG

[0454] GCGAGCCCGGAGTTTCTGCAGCAGGCGCAACCTCTCCTAGAAACGGAGGCCCCGGGGGAGCTGGAG

[0455] G

[0456] Right Mobile element enzyme ITR (SEQ ID NO: 14)

[0457] AATTATTTATGTACTGAATAGATAAAAAAATGTCTGTGATTGAATAAATTTTCATTTTTTACACAAGAAAC

[0458] CGAAAATTTCATTTCAATCGAACCCATACTTCAAAAGATATAGGCATTTTAAACTAACTCTGATTTTGCG

[0459] CGGGAAACCTAAATAATTGCCCGCGCCATCTTATATTTTGGCGGGAAATTCACCCGACACCGTAGTGT

[0460] TAA

[0461] (2) VCD-0031 -D2 mCMV-hABCA4 (NP-pSFK-mCMV_HBI-Optimized hABCA4)

[0462] Left Mobile element enzyme ITR (SEQ ID NO: 2)

[0463] TTAACACTTGGATTGCGGGAAACGAGTTAAGTCGGCTCGCGTGAATTGCGCGTACTCCGCGGGAGCC

[0464] GTCTTAACTCGGTTCATATAGATTTGCGGTGGAGTGCGGGAAACGTGTAAACTCGGGCCGATTGTAAC

[0465] TGCGTATTACCAAATATTTGTT

[0466] Modified hCMV IE promoter (SEQ ID NO: 15)

[0467] GTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAAT

[0468] AATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACG

[0469] GTAAACTGCCTGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTC

[0470] AATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTG

[0471] ACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTC

[0472] AGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCT CCGCGGCCGGGAACGGTGCATTGGAACG

[0473] Spacer 1

[0474] GGATCCGA

[0475] Human Beta-globin Intron II (SEQ ID NO: 16)

[0476] GTGAGTCTATGGGACGCTTGATGTTTTCTTTCCCCTTCTTTTCTATGGTTAAGTTCATGTCATAGGAAG

[0477] GGGATAAGTAACAGGGTACAGTTTAGAATGGGAAACAGACGAATGATTGCATCAGTGTGGAAGTCTCA GGATCGTTTTAGTTTCTTTTATTTGCTGTTCATAACAATTGTTTTCTTTTGTTTAATTCTTGCTTTCTTTTT

[0478] TTTTCTTCTCCGCAATTTTTACTATTATACTTAATGCCTTAACATTGTGTATAACAAAAGGAAATATCTCT

[0479] GAGATACATTAAGTAACTTAAAAAAAAACTTTACACAGTCTGCCTAGTACATTACTATTTGGAATATATG

[0480] TGTGCTTATTTGCATATTCATAATCTCCCTACTTTATTTTCTTTTATTTTTAATTGATACATAATCATTATA

[0481] CATATTTATGGGTTAAAGTGTAATGTTTTAATATGTGTACACATATTGACCAAATCAGGGTAATTTTGCA

[0482] TTTGTAATTTTAAAAAATGCTTTCTTCTTTTAATATACTTTTTTGTTTATCTTATTTCTAATACTTTCCCTAA

[0483] TCTCTTTCTTTCAGGGCAATAATGATACAATGTATCATGCCTCTTTGCACCATTCTAAAGAATAACAGTG

[0484] ATAATTTCTGGGTTAAGGCAATAGCAATATCTCTGCATATAAATATTTCTGCATATAAATTGTAACTGAT

[0485] GTAAGAGGTTTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGA

[0486] TAAGGCTGGATTATTCTGAGTCCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCC

[0487] CACAG

[0488] Spacer 2 (including partial Kozak Sequence) (SEQ ID NO: 17)

[0489] ATCTGTTCCAACCACGGTCACGCTTCGGTGGCCACC hABCA4_optimized_FY_060523 (SEQ ID NO: 18)

[0490] ATGGGCTTCGTGCGCCAGATCCAACTGCTGCTGTGGAAAAACTGGACCTTACGGAAGAGACAGAAGA

[0491] TCAGATTCGTGGTTGAGCTGGTGTGGCCACTGTCTCTCTTCCTGGTGCTGATCTGGCTGAGAAACGCC

[0492] AACCCCCTCTACAGCCATCACGAGTGCCACTTCCCTAACAAGGCTATGCCCAGCGCCGGCATGCTGC

[0493] CCTGGCTGCAGGGAATcTTTTGCAACGTGAATAATCCTTGCTTCCAAAGCCCTACTCCTGGCGAgAGCC

[0494] CAGGCATCGTGAGCAAcTACAACAACAGCATTCTGGCGAGAGTGTACAGAGACTTCCAGGAACTGCTG

[0495] ATGAACGCCCCTGAGTCCCAGCACTTAGGCAGGATCTGGACCGAGCTGCACATCCTGTCCCAGTTTAT

[0496] GGACACCCTGAGGACCCACCCTGAACGGATCGCAGGCAGGGGCATCAGAATCCGCGATATCCTGAAA

[0497] GATGAGGAAACCCTGACCCTGTTCCTGATCAAGAATATCGGACTTAGCGATAGCGTGGTGTACTTACT

[0498] AATCAACAGCCAAGTGCGGCCCGAGCAGTTCGCCCACGGCGTGCCCGATCTGGCTCTGAAGGACATC

[0499] GCCTGTTCCGAGGCACTCTTGGAGCGCTTCATCATCTTCAGTCAGAGAAGAGGCGCCAAGACAGTGC

[0500] GGTACGCCCTTTGTTCACTGTCCCAGGGCACCCTTCAGTGGATCGAGGATACCCTGTACGCCAACGT

[0501] GGACTTCTTcAAGCTGTTCCGTGTGCTGCCTACACTGCTGGATTCAAGAAGCCAAGGCATCAACCTGA

[0502] GAAGCTGGGGTGGTATCTTGTCCGATATGAGCCCTAGAATCCAGGAGTTCATCCACAGACCTAGCATG

[0503] CAAGATCTCCTGTGGGTGACAAGGCCCCTGATGCAGAATGGCGGACCCGAGACATTCACGAAGCTGA

[0504] TGGGAATCCTGTCGGACCTATTGTGCGGATACCCTGAAGGAGGGGGTTCCAGAGTGCTGAGCTTcAA

[0505] CTGGTATGAGGACAACAACTACAAAGCCTTTTTGGGCATCGACAGCACTAGGAAGGATCCTATCTACA

[0506] GCTACGACAGAAGAACCACCTCATTCTGCAACGCCCTGATCCAAAGCCTGGAAAGCAATCCTCTTACA

[0507] AAGATCGCCTGGAGAGCTGCTAAGCCTCTGCTGATGGGCAAGATCCTCTACACCCCAGATTCCCCTG

[0508] CCGCTAGAAGAATCCTGAAGAACGCCAACAGCACCTTCGAGGAGCTGGAGCACGTTCGAAAGTTGGT

[0509] GAAAGCCTGGGAGGAAGTGGGCCCTCAGATCTGGTACTTCTTCGACAACAGCACGCAGATGAACATG

[0510] ATCAGAGACACCCTGGGTAATCCTACCGTCAAGGATTTTCTCAACAGACAGCTGGGAGAAGAGGGCAT

[0511] CACCGCCGAAGCCATCCTCAAcTTTCTGTACAAGGGACCCCGAGAAAGCCAGGCGGACGATATGGCC

[0512] AAcTTCGACTGGCGGGACATCTTCAACATCACAGATAGAACCCTGAGACTGGTGAACCAGTACCTGGA

[0513] ATGTTTGGTGTTGGATAAGTTCGAAAGCTATAACGACGAGACACAGTTGACTCAGCGGGCCCTGTCTC

[0514] TCCTGGAAGAgAACATGTTCTGGGCCGGGGTCGTATTCCCTGATATGTATCCCTGGACCAGTTCTCTG CCACCTCACGTGAAGTACAAGATCCGTATGGACATCGACGTGGTGGAgAAgACAAACAAGATCAAGGA

[0515] TAGATACTGGGACAGCGGCCCACGGGCCGACCCTGTGGAAGACTTCAGATACATCTGGGGAGGATTT

[0516] GCTTACCTGCAAGACATGGTCGAACAGGGCATCACCCGGTCTCAGGTCCAGGCCGAGGCCCCTGTG

[0517] GGAATCTACCTGCAGCAGATGCCTTACCCGTGCTTCGTGGATGACTCCTTCATGATCATCCTGAATAG

[0518] ATGCTTCCCTATCTTCATGGTGCTGGCCTGGATCTACAGCGTGAGCATGACCGTCAAGTCCATCGTCC

[0519] TGGAAAAGGAACTGAGATTGAAAGAGACCCTGAAGAACCAGGGCGTGAGCAACGCCGTAATCTGGTG

[0520] CACTTGGTTCCTGGATTCTTTCAGCATAATGAGCATGAGCATCTTCCTGCTGACCATCTTCATTATGCA

[0521] CGGCAGAATCCTGCATTATTCCGACCCGTTCATCCTGTTCCTGTTCCTGCTGGCCTTCAGCACCGCTA

[0522] CCATCATGCTGTGCTTCTTGCTGAGTACATTCTTCAGCAAGGCCAGCCTCGCCGCCGCCTGCAGCGG

[0523] CGTGATCTACTTCACCCTGTACCTGCCCCACATTCTTTGCTTCGCCTGGCAAGACAGAATGACAGCTG

[0524] AACTGAAGAAGGCCGTGTCCCTGCTGAGCCCCGTGGCCTTCGGCTTCGGCACCGAGTATCTTGTGAG

[0525] ATTTGAGGAGCAGGGCCTGGGCCTGCAGTGGAGCAACATCGGAAAcTCACCCACAGAGGGCGACGA

[0526] GTTCAGCTTTTTGCTGAGTATGCAGATGATGCTGCTGGACGCCGCTGTGTACGGCCTGCTCGCATGGT

[0527] ACCTGGACCAGGTCTTTCCGGGCGACTACGGCACCCCCCTGCCTTGGTACTTTCTCCTGCAGGAGTC

[0528] TTACTGGCTGGGTGGAGAAGGCTGCAGCACCCGGGAGGAGAGAGCACTCGAAAAAACAGAGCCTCT

[0529] GACAGAGGAAACCGAAGATCCCGAGCACCCCGAAGGCATCCACGACTCTTTCTTcGAGAGAGAACAC

[0530] CCCGGCTGGGTGCCAGGAGTGTGTGTGAAgAACCTGGTgAAGATCTTTGAGCCCTGCGGCAGACCAG

[0531] CCGTGGACCGGCTgAACATCACCTTCTACGAGAACCAAATCACCGCCTTCCTGGGCCACAACGGCGC

[0532] CGGCAAGACCACGACCCTGTCCATCCTGACCGGCCTGCTGCCTCCTACAAGCGGGACTGTGCTCGTC

[0533] GGAGGCAGAGACATCGAGACATCTCTGGACGCCGTGAGACAGTCACTAGGCATGTGCCCTCAACAcA

[0534] ATATCCTATTCCACCACCTGACAGTAGCAGAACACATGCTGTTCTACGCCCAGCTGAAGGGAAAGAGC

[0535] CAGGAGGAAGCCCAGCTGGAGATGGAAGCTATGCTCGAGGATACCGGACTGCACCATAAGCGAAATG

[0536] AGGAAGCCCAGGACCTGTCCGGCGGAATGCAGAGAAAGCTGTCTGTGGCCATAGCCTTCGTAGGGG

[0537] ACGCCAAGGTGGTGATCCTGGACGAGCCCACCTCTGGCGTGGACCCTTACTCGCGGAGATCAATATG

[0538] GGACCTGCTCCTGAAGTACAGGTCGGGCAGAACCATCATCATGTCCACACACCACATGGACGAGGCC

[0539] GATCTGCTAGGCGACCGGATCGCCATTATCGCACAGGGACGACTGTACTGTAGTGGCACACCTCTGT

[0540] TCCTGAAGAACTGCTTTGGCACAGGCCTGTACCTCACCCTGGTGAGAAAAATGAAgAATATCCAAAGC

[0541] CAAAGGAAAGGCTCTGAGGGAACCTGTAGCTGTAGCAGCAAGGGCTTTAGCACCACCTGCCCTGCCC

[0542] ATGTGGATGACCTAACACCTGAGCAGGTGCTGGACGGCGATGTGAACGAGCTGATGGATGTGGTGCT

[0543] CCACCACGTGCCGGAAGCCAAACTAGTGGAATGCATCGGCCAGGAGCTGATCTTCCTGCTGCCAAAC

[0544] AAGAAcTTCAAGCACCGGGCCTACGCCAGCCTGTTCAGAGAGCTGGAAGAGACATTGGCAGATCTGG

[0545] GACTGAGCTCTTTCGGCATCAGCGATACCCCGCTGGAGGAAATcTTTCTCAAGGTGACGGAGGATAGC

[0546] GATAGCGGCCCTCTGTTCGCCGGCGGCGCCCAACAGAAGCGTGAGAACGTGAACCCCAGACACCCA

[0547] TGCCTGGGCCCCCGGGAgAAAGCGGGGCAGACCCCTCAAGACAGCAACGTCTGCTCTCCGGGcGCC

[0548] CCTGCcGCTCAcCCTGAGGGGCAGCCTCCCCCCGAGCCAGAGTGTCCTGGACCACAGCTGAATACCG

[0549] GCACACAACTGGTGCTCCAACACGTTCAGGCCCTGCTGGTGAAGCGGTTCCAGCACACCATCAGGAG

[0550] CCACAAAGATTTCCTGGCCCAGATCGTGTTGCCCGCCACGTTCGTGTTCCTGGCACTGATGCTGTCCA

[0551] TCGTGATCCCTCCTTTCGGCGAGTATCCTGCCCTGACCCTGCACCCTTGGATCTATGGCCAGCAGTAC

[0552] ACCTTcTTCTCAATGGATGAACCTGGCAGCGAGCAGTTTACAGTGCTGGCTGACGTGCTGCTGAACAA

[0553] GCCCGGCTTCGGAAACCGGTGCCTGAAGGAAGGCTGGCTGCCCGAGTACCCTTGCGGCAACAGCAC

[0554] ACCTTGGAAGACCCCTAGCGTCAGTCCTAACATCACACAGCTGTTCCAGAAGCAGAAGTGGACACAAG

[0555] TgAATCCCTCCCCTAGTTGCCGGTGTAGCACACGGGAGAAGCTGACCATGCTGCCCGAATGCCCTGA

[0556] GGGCGCCGGTGGTCTGCCGCCTCCTCAGAGAACCCAACGTAGTACCGAGATCCTGCAGGACCTCAC

[0557] CGACAGAAACATCAGCGACTTCCTTGTTAAGACCTACCCCGCCctgATTCGGAGCAGCCTGAAGAGCAA

[0558] GTTCTGGGTGAACGAACAGAGATATGGCGGCATCTCTATCGGCGGCAAgTTGCCTGTCGTGCCCATTA

[0559] CCGGCGAGGCACTGGTGGGCTTCCTGAGCGACCTGGGCCGTATTATGAACGTGAGCGGTGGTCCTAT

[0560] CACAAGAGAGGCCTCTAAAGAGATCCCTGACTTCCTGAAGCACCTGGAGACAGAGGATAACATCAAG GTGTGGTTcAACAACAAGGGCTGGCACGCCCTAGTCTCGTTCTTGAACGTGGCACACAACGCCATTCT

[0561] GCGGGCCTCTCTGCCTAAGGATAGATCCCCTGAAGAGTACGGCATCACGGTGATCAGCCAACCTCTG

[0562] AATCTGACCAAGGAGCAGCTGAGCGAGATCACTGTGTTGACCACAAGCGTGGACGCCGTCGTGGCTA

[0563] TCTGCGTGATCTTCAGTATGAGCTTCGTGCCCGCCAGCTTCGTGCTGTACCTGATCCAGGAGAGAGTG

[0564] AACAAGAGCAAACACCTGCAGTTCATCTCTGGGGTTAGCCCTACAACCTACTGGGTCACCAATTTCTTA

[0565] TGGGATATCATGAACTACAGCGTATCCGCCGGACTGGTAGTGGGCATTTTCATTGGCTTCCAGAAAAA

[0566] GGCCTACACGTCTCCTGAAAATCTGCCCGCTCTGGTAGCTCTACTCCTGCTGTACGGCTGGGCCGTG

[0567] ATCCCTATGATGTACCCTGCCAGTTTTCTATTCGACGTGCCCAGCACCGCCTACGTGGCCTTATCCTG

[0568] CGCCAACCTGTTTATCGGCATCAACAGCAGCGCCATCACTTTCATTCTCGAGCTGTTCGAGAACAACC

[0569] GGACACTGCTGAGATTCAACGCAGTCCTTCGGAAGCTGCTGATCGTGTTTCCTCACTTTTGTCTGGGC

[0570] CGTGGCCTGATCGACCTGGCTCTGTCTCAGGCAGTGACCGATGTGTACGCCAGATTCGGCGAAGAGC

[0571] ACTCTGCCAACCCCTTCCACTGGGACCTGATCGGcAAAAATCTGTTCGCCATGGTGGTGGAGGGTGTG

[0572] GTTTATTTCCTCCTGACACTGCTGGTGCAGAGACACTTCTTCTTGAGCCAGTGGATCGCCGAACCAAC

[0573] AAAGGAACCTATCGTGGACGAAGATGATGATGTGGCTGAAGAGAGACAGAGAATCATCACTGGCGGT

[0574] AAcAAAACTGATATCCTGAGACTGCATGAACTAACAAAGATCTATCCTGGTACCTCTTCACCTGCCGTG

[0575] GACCGGCTTTGTGTGGGAGTTAGGCCTGGCGAGTGCTTCGGCTTGTTGGGCGTGAACGGCGCAGGC

[0576] AAGACAACAACATTCAAGATGCTGACCGGCGACACAACCGTTACCAGCGGCGACGCCACAGTGGCTG

[0577] GAAAGTCTATCCTGACAAACATCAGCGAGGTCCACCAGAACATGGGCTACTGCCCCCAGTTTGACGCC

[0578] ATCGACGAGCTGCTGACCGGCAGAGAACACCTCTACCTCTACGCCCGACTGCGAGGCGTGCCTGCC

[0579] GAAGAGATTGAGAAGGTGGCCAATTGGAGCATCAAGTCCCTGGGCCTCACTGTGTACGCCGATTGCC

[0580] TGGCTGGTACTTACAGCGGCGGCAACAAGCGGAAGTTGAGCACCGCCATTGCCCTAATcGGCTGCCCt

[0581] CCCCTGGTGCTGCTGGACGAGCCTACCACAGGAATGGATCCTCAGGCCAGAAGAATGCTATGGAACG

[0582] TGATCGTATCCATcATTAGAGAGGGCCGGGCCGTGGTGTTGACTTCTCACAGCATGGAAGAATGCGAA

[0583] GCCCTGTGCACCCGCCTGGCGATTATGGTGAAGGGAGCCTTCCGGTGCATGGGAACCATCCAGCACC

[0584] TGAAGTCAAAGTTTGGAGACGGCTACATCGTGACCATGAAAATCAAGTCGCCTAAGGACGACCTGCTC

[0585] CCAGACCTGAACCCTGTGGAGCAGTTCTTCCAGGGCAACTTTCCTGGTTCTGTGCAGAGAGAGCGCC

[0586] ACTACAATATGCTTCAGTTCCAGGTGTCTAGCAGTAGCCTTGCTAGAATCTTCCAGCTCCTGCTGAGCC

[0587] ACAAGGACAGCCTGCTGATTGAGGAATACTCCGTCACCCAGACTACCCTGGATCAGGTCTTCGTGAAC

[0588] TTCGCTAAGCAGCAGACCGAGTCCCACGACCTGCCCCTGCACCCTCGGGCCGCTGGCGCTTCCAGA

[0589] CAGGCCCAGGACTGA

[0590] Spacer 3 (SEQ ID NO: 19)

[0591] TAGTAAACGCGT bGH Poly A (SEQ ID NO: 20)

[0592] GCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCC

[0593] TTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTG

[0594] AGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGAC AATAGCAG Spacer 4 (including restriction site E for our modular cloning backbone) (SEQ ID NO: 21)

[0595] GCATGCTCTAGAACTAGT

[0596] Right Mobile element enzyme ITR (SEQ ID NO: 14) AATTATTTATGTACTGAATAGATAAAAAAATGTCTGTGATTGAATAAATTTTCATTTTTTACACAAGAAAC

[0597] CGAAAATTTCATTTCAATCGAACCCATACTTCAAAAGATATAGGCATTTTAAACTAACTCTGATTTTGCG

[0598] CGGGAAACCTAAATAATTGCCCGCGCCATCTTATATTTTGGCGGGAAATTCACCCGACACCGTAGTGT TAA

[0599] Other constructs for nucleic acids delivery

[0600] Exemplary plasmid DNA useful for the compositions and methods of the present disclosure is listed below herein:

[0601] This invention is further illustrated by the following non-limiting examples. EXAMPLES

[0602] Hereinafter, the present disclosure will be described in further detail with reference to examples. These examples are illustrative purposes only and are not to be construed to limit the scope of the present invention. In addition, various modifications and variations can be made without departing from the technical scope of the present invention. Example 1: Formulation Development FIG. 1 shows formulations load DNA / RNA. Lipid content is given in % mol and Cationic / Total lipid:NA is in mg:mg.

[0603] FIG. 2 shows formulations of MC3 and C12-200 mixed LNPs. Mixed lipids improved %EE (keeping cationic lipid constant) gave excellent results.

[0604] A higher MC3 Cationic lipid : nucleic acid (NA) also allowed for reduction of %PEGs as shown in FIG. 3A and FIG. 3B (showing size, polydispersity index (PDI), and %EE).

[0605] FIG. 4A and FIG. 4B show MC3 compositions also allowed changing PEG anchors of different lengths with high %EE. The composition was 9% DOPE + 45% MC3 + 44% CHOLESTEROL + 2% PEG. PEG studied were DMG and Ceramide-based. Further, C8 and C16, length of lipids conjugated to PEG, were analyzed.

[0606] FIG. 5A and FIG. 5B show effects of different pH on %EE. Different pH buffers had similar profiles (high %EE). The composition was 9% DOPE + 45% MC3 + 44% CHOLESTEROL + 2% PEG.

[0607] These results based on FIGs. 4A-4B and FIGs. 5A-5B demonstrated that varying PEG does not change the LNP size and PDI significantly and encapsulation remains unaffected.

[0608] FIG. 6 shows that SS-OP LNPs can be formulated with robust size and encapsulation. The sizes were very small (<100nm) for all formulations. Nearly all particles resulted in precipitation but high N:P PEG non- MW corrected resulted in few precipitates.

[0609] Example 2: In Vivo Studies with Various Routes of Administration

[0610] FIG. 7 shows the compositions of LNPs with pDNA- nRNA that are suitable for use in subretinal injection. For instance, mice are subretinally injected with these formulations and their characteristics observed, such as images of eyes and / or ocular fluorescence. For instance, transfection of retinal cells via subretinal injection in Ai9 mice is used to confirm delivery.

[0611] Example 3: Formulation Engineering

[0612] Without wishing to be bound by theory, to decrease the toxicity of cationic lipids and ionizable lipids in formulations and to widen the therapeutic window, various amount (mg) of cationic lipids and ionizable lipids were assessed.

[0613] In prior experiments, the amount of cationic lipids and ionizable lipids remained constant in formulations and the toxicity profile did not change. As shown in FIG. 8, additional experiments focused, inter alia, on altering the amount of cationic lipid and ionizable lipid in formulations. These experiments helped, inter alia, assessing C12-200 and led to consider other compositions. The results of these experiments showed that LNP size is dependent on MC3 compositions whereas the PDI and the percentage of encapsulation remain unchanged. Thus, LNP formation depends on ionizable lipid.

[0614] As shown in FIG. 9, additional LNP screenings and new engineering methods were established. The physicochemical characteristics such as DLS size (where DLS relates to dynamic light scattering measuring the hydrodynamic radius of the nanoparticle), PDI (where PDI, polydispersity index, relates to the homogeneity in the size of the particles, lower is more equally sized particles), and percentage of encapsulation were reported for new MC3 LNPs compositions.

[0615] FIG. 10 shows the selection of specific formulation for mRNA delivery with a luciferase reporter. LNPs with dual cargos (mVLDLR + Flue mRNA) were made at various ratios as shown in FIG. 10 and injected into BalbB / C mice. The luciferase signal (total flux) was measured in I VIS at 6hr and 24hrs after injections. The 1-5-5 ratio with the MC3 lipid LNPs showed the highest mRNA delivery with respect to the luciferase signal from Flue mRNA. The MC3 formulation with 1-5-5 composition was selected to be used in various diseases formulations such as and inherited retinal diseases (IRD).

[0616] As shown in FIG. 11, additional experiments were designed for various formulations (ionizable lipids and hydrolysable lipids) to be injected sub-reti nally. In order to narrow LNP screening and to evaluate the safety of various ionizable lipids and hydrolysable lipids at 2 different doses, all other LNP parameters and components were kept consistent.

[0617] FIG. 12 shows LNP tolerability and expression with various SS-OP, and DOTAP / PEG formulations. Fundus images showing vasculature changes 24 hrs post subretinal injection (Top row). The white spots indicated blanching or non-perfused vasculature as a response to the injection. Both SS-OP formulations and DOTAP high PEG formulation were better tolerated compared to DOTAP low PEG formulation that showed significant blanching. Additionally, the wavy pattern was indicative of significant toxicity response and cell death. GFP fluorescence images indicating potential GFP mRNA protein signal from LNPs (Bottom Row). Blanching can lead to confounding results as non-perfused and non-viable photoreceptors and RPEs also showed autofluorescence. As used herein, the term blanching refers to focal areas of pure white in the treated area, most likely due to areas of non-perfused vasculature. While it is unclear if the blanching correlates with OCT thinning in mice but overall, it is not a positive predictor of overall tolerability.

[0618] The level of cell death and tolerability with various SS-OP, and DOTAP / PEG formulations are shown in FIG.

[0619] 13. Post subretinal injection of mRNA only LNPs, in-life OCT images were taken at various timepoints (at 21 days). The brackets encompass the outer nuclear layer (ONL) and the subsequent degradation / thinning of the ONL is indicative of cell death and lack of tolerability. A qualitative scoring system was established by comparing ONL degenerating at the site of injection versus the opposite side of the same eye. A score of 0 indicated < 10% ONL degeneration (seen with AAV samples), 1 indicated > 10% and < 50% degeneration, and 2 indicated > 50% degeneration of ONL. A score of 3 indicated > 50% ONL degeneration that extended beyond the treated area. The eyes were scored, averaged, and graphed against LNPs that were used in the experiment. As illustrated in FIG. 13, SS-OP and DOTAP LNPs showed low level of degeneration. Bimodal distribution with low number was expected and the red dotted line indicated level of tolerability score below which LNPs should be tolerable for higher species and DNA.

[0620] As shown in FIG. 14, SS-OP and DOTAP / PEG formulations provided high level of mRNA expression with low toxicity. After extraction of the eyes post takedown, neural retina was separated from the eyecup under a microscope. The isolated tissue was disrupted and purified for GFP ELISA. An Off-the-shelf GFP ELISA kit was utilized to evaluate the concentration of GFP protein produced from the mRNA per tissue. Based on the total protein extracted and the ratio of GFP to total protein from BOA assay and ELISA, GFP / tissue was calculated and is shown on the y-axis of FIG. 14. Given the log-scale, SS-OP and SS-EC formulations showed a 1-2 log increase in GFP protein compared to the baseline (PBS) and DOTAP 5% PEG formulation showed 2 log increase. DOTAP 0.5% PEG formulation showed a bimodal distribution indicating that mRNA expression was significantly lower except for one eye. No significant difference was detected with GFP present in RPE vs. Retina from a biodistribution perspective. As shown, SS-OP and SS-EC LNPs were best tolerated followed by DOTAP. These results showed that SS-OP, SS-EC, and DOTAP LNPs are potential candidates for DNA delivery with low toxicity. The lipid formulations 1-37-5.2 indicated the absolute molar helper-SS-OP-Cholesterol ratio, the 1-1-2 formulation indicated the absolute molar helper-SS-EC-cholesterol ratio, and the 13:1 formulation indicated the DOTAP:total nucleic acid w / w ratio.

[0621] As shown in FIG. 15, further experiments were designed for other various formulations (ionizable lipids and hydrolysable lipids) to be injected sub-reti nally. In order to narrow LNP screening and to evaluate the safety of various ionizable lipids and hydrolysable lipids at 2 different doses, all other LNP parameters and components were kept consistent.

[0622] FIG. 15 and FIG. 16 show LNP tolerability and expression with various SS-OP, SS-EC, and DOTAP formulations. In particular, a strong fundus expression was observed with SS-OP and SS-OP 2. Fundus images showed vasculature changes 24 hrs post subretinal injection (top row). The white spots indicated blanching or non-perfused vasculature as a response to the injection. Both SS-OP formulations and DOTAP high PEG formulation were better tolerated as compared to DOTAP with low PEG formulation that showed significant blanching. Additionally, the wavy pattern indicated significant toxicity response and cell death. GFP fluorescence images indicating potential GFP mRNA protein signal from LNPs (bottom row). Blanching can lead to confounding results as non-perfused and non-viable photoreceptors and RPEs also showed autofluorescence.

[0623] FIG. 18 and FIG. 19 showthe level of cell death and tolerability with various SS-OP, SS-EC, and DOTAP / PEG formulations. Post subretinal injection of mRNA only LNPs, in-life OCT images were taken at various timepoints (at 21 days). The brackets encompass the ONL and the subsequent degradation / thinning of the outer nuclear layer indicated cell death and lack of tolerability. A qualitative scoring system was established by comparing ONL degenerating at the site of injection versus the opposite side of the same eye. A score of 0 indicated < 10% ONL degeneration (seen with AAV samples), 1 indicated > 10% and < 50% degeneration, and 2 indicated more than 50% degeneration of ONL. A score of 3 indicated > 50% ONL degeneration that extended beyond the treated area. The eyes were scored, averaged, and graphed against LNPs that were used in the experiments. As shown, SS-OP and SS-EC LNPs were best tolerated followed by DOTAP being least tolerated and having the highest score. These results indicated that SS-OP, SS-EC, and DOTAP LNPs are potential candidates for DNA delivery with low toxicity. The lipid formulations 1-3.7-5.2 indicated the absolute molar helper-SS-OP-Cholesterol ratio, the 1-1-2 formulation indicated the absolute molar helper- SS-EC-cholesterol ratio, and the 13:1 formulation indicated DOTAP:total nucleic acid w / w ratio.

[0624] As shown in FIGS. 20A-20B, screening of LNPs with immuno-silent mRNA cargo (GFP) allowed selection of lipids with a favourable tolerability profile in vivo. The tolerability was evaluated at 3 days post injection by optical coherence tomography (OCT) imaging of the retina in-life which included the treated area and the opposite side (non-injected) (FIG. 20A). The thickness of the ONL which contains photoreceptors is indicative of the tolerability of the LNP formulation. As photoreceptor cell death occurs, the ONL thins, which was qualitatively demonstrated via a scoring system where (i) a score of 0 represents less than 10% loss of thickness in ONL compared to the non-injected region; (ii) a score of 1 represents 10-50% loss of ONL thickness compared to the non-injected region; and (iii) a score of 2 represents more than 50% loss of ONL thickness in the treated area compared to the non-injected region. As shown in FIG. 20B, the disclosed LNPs showed minimal degeneration compared to standard LNPs. Graphing the ONL degeneration from the study showed that the SS-OP formulations (i.e., the disclosed LNP 1 : SS-OP (13:1 SS-OP:NA, 1 :3.7:5.3 %mol DOPE:% mol SS-OP: %mol Cholesterol; 5% PEG)) containing GFP mRNA only are best tolerated in vivo compared to SSEC and ALC-0315 formulations. The degeneration score indicated that lipids similar to ALC- 0315 are not tolerated for mRNA and thus are not of interest for DNA, however, the use of SS-OP formulation is suitable for dual cargo delivery.

[0625] Example 4: Cargo Delivery to Neural Retina Cells

[0626] As shown in FIGS. 21A-21 B subretinal (SR) injection of SS-OP / mRNA was delivered to neural retina cells, which comprise many cell types, including photoreceptors (PRs), and to retinal pigment epithelial (RPE) cells. In particular, Cre reporter mRNA with SS-OP LNPs (36.3% SS-OP, 50.7% Cholesterol, 9.8% DOPE, 5% PEG) was delivered through subretinal injection to about 60% of mouse neural retina cells within the treated area. As expected, Cre reporter mRNA expression was limited to the treated area indicating substantial delivery of SS-OP LNPs into the retina (FIG. 21A) with a delivery and expression of mRNA into all 3 cell types, of which the inner and outer nuclear layers (INL, ONL) contain photoreceptors.

[0627] When neural retina cells were transfected with the Cre reporter mRNA / SS-OP LNP, a dose response was detected with 0.2 ug / eye of Cre reporter mRNA having the highest % transfection (-60%) as compared to the other doses (FIG. 21 B).

[0628] As shown in FIG. 22, SS-OP / mRNA can be delivered into nonhuman primates (NHPs) photoreceptors (PRs) and retinal pigment epithelial (RPE) cells. Subretinal (SR) injection of GFP reporter mRNA SS-OP LNPs (36.3% SS-OP, 50.7% Cholesterol, 9.8% DOPE, 5% PEG) were performed in NHPs with formulations made in aseptic conditions with 75 nm in size and less than 0.2 PDI. 24 hrs post-injections, the expression decreased to zero outside the treated area, representative of LNP based expression in vivo.

[0629] Constructs for ABCA4 nucleic acids delivery

[0630] ATP binding cassette subfamily A member 4 (ABCA4) protein is a retina-specific ABC transporter with N- retinylidene-PE as a substrate. It is expressed exclusively in retina photoreceptor cells, and the gene product mediates transport of an essential molecule, all-trans-reti nal aldehyde (atRAL), across the photoreceptor cell membrane. Mutations in this gene are found in patients diagnosed with Stargardt disease, a form of juvenileonset macular degeneration. Mutations in this gene are also associated with other conditions such as retinitis pigmentosa-19, cone-rod dystrophy type 3, early-onset severe retinal dystrophy, fundus flavimaculatus, and macular degeneration age-related 2. To assess the therapeutic window for SS-OPIABCA4 DNA delivery, GFP vs. Abca4 were used as cargo within SS-OP LNPs (36.3% SS-OP, 50.7% Cholesterol, 9.8% DOPE, 5% PEG) and the current therapeutic index was evaluated (FIG. 23). OCT measurements were taken at day 12 (D12) of the mice injected sub- retinally with the LNPs. Based on the differences in cargo base pair size, the amount of abca4 or GFP was modified to match the same nM of nucleic acid. 7 nM was shown to be the therapeutic index for the DNA regardless of the cargo size with an OCT degeneration score lower than 1 on average. The degeneration score indicated that tolerability is driven by the number of molecules of DNA delivery, regardless of the DNA size or sequence.

[0631] As shown in FIGS. 24A-24B, the SS-OP / ABCA4 delivery to photoreceptors (PRs) and retinal pigment epithelial (RPE) cells following subretinal (SR) injection was within current therapeutic index (Tl). For 7 nM of DNA (25 ng for GFP and 50 ng for Abca4), 0.01 copies / cell for neural retina and 1 copy / cell for RPE were detected for both Abca4 and GFP. Overall, abca4 DNA delivery was lowered compared to GFP. The disclosed formulations deliver preferentially to the RPE, with lower efficiency in the neural retina. Quantifying DNA delivery and the generated transcript (FIG. 24A). As shown in FIG. 24B, both Abca4 and GFP have the same level of fold change indicating increased levels of mRNA produced from the DNA. Thus, LNP delivery of Abca4 was productive and led to transcription of mRNA as well.

[0632] As shown in FIGS. 25A-25B, following subretinal (SR) delivery of AAV5 with LNP / mobile element enzyme (SEQ ID NO: 1), neural retina integration was detected. AAV was used as a tool for DNA delivery and to evaluate mobile element enzyme (SEQ ID NO: 1) integration. Dual subretinal delivery was evaluated into mice staggered by 21 days (AAV followed by LNP with Cre mRNA-miobile element enzyme (SEQ ID NO: 1) mRNA (36.3% SS-OP, 50.7% Cholesterol, 9.8% DOPE, 5% PEG)). A widespread expression by the Cre LNP was detected and in the dual injected groups, a large population of cells received dual mRNA leading to dual protein expression (FIG. 25A). It was concluded that cells that received Cre mRNA and showed a positive expression, also received mobile element enzyme (SEQ ID NO: 1) mRNA given that they were coencapsulated in the same LNP.

[0633] The copy number per cell via ddPCR of cells in the neural retina (-80% photoreceptors) that express both GFP from AAV and cre / mobile element enzyme (SEQ ID NO: 1) from LNP was assessed (FIG. 25B). All doses, with 0.05 ug / ml being the highest show an increase in DNA copy number compared to AAV alone was indicative of integration via mobile element enzyme (SEQ ID NO: 1) (FIG. 25B). Increased levels of mobile element enzyme (SEQ ID NO: 1) showed low tolerability, as demonstrated by a decrease in DNA copy number.

[0634] Example 5: Successful in vivo ABCA4 gene replacement in a mouse model of Stargardt disease using a mammalian transposon system

[0635] An engineered mammalian transposase was co-encapsulated in a nanoparticle to integrate ABCA4 in the genome (e.g. by using construct (1) and / or (2) as provided elsewhere above herein).

[0636] DNA and RNA dosing with 7nM (low dose, LD) nucleic acid and 13nM (high dose, HD) nucleic acid.

[0637] Transcript production was analyzed in ARPE19, HEK293T cells, and Abca4- / - mice using ABCA4+ fluorescence-activated cell sorting (FACS), droplet digital polymerase chain reaction (ddPCR), and quantitative reverse transcription (RTqPCR).

[0638] To assess dosing, tolerability, and delivery in vivo subretinal injections were performed in 24 mice (1 uL per eye) in three groups of Abca4- / - mouse eyes: vehicle alone (n=8) and treated groups with LD (n=8) and HD (n=8). The mice were analyzed by serial fundoscopy, optical coherence tomography (OCT), ecto-5'- nucleotidase (CD73+) / ABCA4+ photoreceptor sorting of dissected neural retina by FACS, histology, immunohistochemistry (IHC), ddPCR, and RT-qPCR analyses.

[0639] FACS analysis identified a single ABCA4+ and dual ABCA4+ / FLAG+ in ARPE19 and HEK293T cell populations. Following confirmation of genomic integration, both treated Abca4- / mouse groups showed ABCA4 expression by FACS (P<0.05) in 10% of all retinal photoreceptors with no difference (P>0.1 ) in OCT retinal layer measurements between the vehicle alone and treated groups.

[0640] As shown in FIGS. 26A-26B, the delivery of CAG-hABCA4 + Mobile element enzyme (SEQ ID NO: 1 ) mRNA into photoreceptors (PRs) exhibited a good tolerability with a SS-OP LNP (36.3% SS-OP, 50.7% Cholesterol, 9.8% DOPE, 5% PEG)). In particular, minimal to a no degeneration was observed with SS- OP / ABCA4-miobile element enzyme (SEQ ID NO: 1) delivery to the retina (FIG. 26A). The OCT degeneration score of the ONL was within the tolerable range for LNPs for both doses. Compared to the background (2.7%) both doses of LNP indicated an increase in Abca4 expression of more than 5% (FIG. 26B).

[0641] As shown in FIG. 27 SS-OP / ABCA4 and mobile element enzyme (SEQ ID NO: 1) are expressed in mouse photoreceptors (PRs). Immunohistochemistry (IHC) assays showed that the Abca4 was localized to the outer segments of the photoreceptors which themselves were naturally expressed as a transport protein. The localization showed that the LNP was able to deliver the Abca4 DNA and the DNA was able to be translated into protein that is expressed in the OS of the photoreceptors. Thus, Retinal IHC proved precise restoration of the ABCA4 protein in its natural photoreceptor OS location. ABCA4 ddPCR and RTqPCR confirmed these findings.

[0642] The results supported the feasibility and tolerability of subretinal administration of a non-viral, integrative gene therapy to deliver a large gene such as ABCA4 to photoreceptors in a mouse model of STGD1 .

[0643] EQUIVALENTS

[0644] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features herein set forth and as follows in the scope of the appended claims.

[0645] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

[0646] INCORPORATION BY REFERENCE

[0647] All patents and publications referenced herein are hereby incorporated by reference in their entireties.

[0648] The publications discussed herein are provided solely fortheir disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

[0649] As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections.

Claims

CLAIMSWhat is claimed is:1 . A composition comprising:(a) one or more nucleic acid-based agents, and(b) one or more lipids, the one or more lipids comprising:(i) a delivery lipid,(ii) cholesterol,(iii) a helper lipid, and(iv) optionally, a PEGylated lipid.

2. The composition of claim 1 , wherein the delivery lipid is selected from MC3, SS-OP, SS_EC, and any combination thereof.

3. The composition of claim 1 or 2, wherein the helper lipid is selected from distearoylphosphatidylcholine (DSPC), 1 ,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), and any combination thereof.

4. The composition of any one of claims 1-3, wherein the PEGylated lipid is selected from PEG-2K-DMG, C8 PEG 2K, and C16 PEG 2K.

5. The composition of any one of claims 1-4, comprising:(i) a delivery lipid comprising from about 15 mol % to about 75 mol % of the total lipid present,(ii) cholesterol comprising from about 25 mol % to about 65 mol % of the total lipid present,(iii) a helper lipid comprising from about 5 mol % to about 40 mol % of the total lipid present, and(iv) a PEGylated lipid comprising from about 0.5 mol % to about 7 mol % of the total lipid present.

6. The composition of any one of claims 1-5, wherein the one or more lipids comprises:(i) a delivery lipid comprising from about 30 mol % to about 60 mol % of the total lipid present, the delivery lipid being MC3,(ii) cholesterol comprising from about 30 mol % to about 60 mol % of the total lipid present,(iii) a helper lipid comprising from about 5 mol % to about 35 mol % of the total lipid present, optionally being one or more of:DSPC from about 8 mol % to about 12 mol % of the total lipid present, DOPC from about 8 mol % to about 10 mol % of the total lipid present, and DOPE from about 7.5 mol % to about 25 mol % of the total lipid present, and(iv) a PEGylated lipid comprising from about 0.5 mol % to about 3 mol % of the total lipid present, optionally being one or more of:PEG-2K-DMG from about about 0.5 mol % to about 3 mol % of the total lipid present,C8 PEG 2K ceramide from about about 0.5 mol % to about 2.5 mol % of the total lipid present,C16 PEG 2K ceramide from about about 0.5 mol % to about 2.5 mol % of the total lipid present.

7. The composition of any one of claims 1-5, wherein the one or more lipids comprises:(i) a delivery lipid comprising from about 25 mol % to about 75 mol % of the total lipid present, the delivery lipid being SS-OP,(ii) cholesterol comprising from about 25 mol % to about 65 mol % of the total lipid present,(iii) a helper lipid comprising from about 5 mol % to about 35 mol % of the total lipid present, optionally being one or more of:DOPC from about 5 mol % to about 35 mol % of the total lipid present, andDOPE from about 2.5 mol % to about 30 mol % of the total lipid present, and(iv) a PEGylated lipid comprising from about 0.5 mol % to about 5 mol % of the total lipid present, optionally being PEG-2K-DMG.

8. The composition of any one of claims 1-5, wherein the one or more lipids comprises:(i) a delivery lipid comprising from about 15 mol % to about 35 mol % of the total lipid present, the delivery lipid being SS-EC,(ii) cholesterol comprising from about 35 mol % to about 60 mol % of the total lipid present,(iii) a helper lipid comprising from about 15 mol % to about 35 mol % of the total lipid present, and(iv) a PEGylated lipid comprising from about 3 mol % to about 7 mol % of the total lipid present, optionally being PEG-2K-DMG.

9. The composition of any one of claims 1 -8, wherein the composition has a weight to weight ratio of delivery lipid to one or more nucleic acid-based agents of about 5:1 to about 15:1 , or about 7:1 to about 13:1 , or about 8:1 to about 12:1, or about 5:1, or about 6:1 , or about 7: 1 , or about 8:1 , or about 9:1, or about 10:1 , or about 11 :1 , or about 12:1 , or about 13:1 , or about 14:1, or about 15:1.

10. The composition of any one of claims 1-9, wherein the composition has a weight to weight ratio of total lipid to one or more nucleic acid-based agents of about 10:1 to about 35:1 , or about 15:1 to about 30:1, or about 20:1 to about 25:1 , or about 15:1 , or about 20:1 , or about 25:1, or about 30:1.

11. The composition of any one of claims 1-10, wherein the composition forms a nucleic acid / lipid particle.

12. The composition of any one of claims 1-11 , wherein the one or more lipids associate into a lipid nanoparticle (LNP).

13. The composition of claim 1-12, wherein the LNP encapsulates the one or more nucleic acid-based agents.

14. The composition of any one of claims 1-13, wherein the one or more nucleic acid-based agents comprise a nucleic acid which encodes a mobile element enzyme.

15. The composition of claim 1-14, wherein the mobile element enzyme has the amino acid sequence of SEQ ID NO: 1 , or an amino acid sequence having at least about 80%, or an amino acid sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.

16. The method of any one of claims 1-15, wherein the mobile element enzyme comprises an amino acid other than serine at the position corresponding to position 2 of SEQ ID NO: 1 .

17. The method of any one of claims 1-16, wherein the amino acid is a non-polar aliphatic amino acid, optionally a non-polar aliphatic amino acid optionally selected from G, A, V, L, I, and P, optionally A.

18. The method of any one of claims 1-17, wherein the mobile element enzyme does not have additional residues at the C terminus relative to SEQ ID NO: 1 .

19. The method of any one of claims 1-18, wherein the enzyme has one or more mutations which confer hyperactivity.

20. The method of any one of claims 1-19, wherein the enzyme has one or more amino acid substitutions selected from S8X1, C13X2, and N125Xs, or positions corresponding thereto relative to SEQ ID NO: 1.

21. The method of claim 1-20, wherein the enzyme has S8X1 substitution, or at a position corresponding thereto relative to SEQ ID NO: 1 .

22. The method of claim 1-21, wherein the enzyme has C13X2 substitution, or at a position corresponding thereto relative to SEQ ID NO: 1 .

23. The method of claim 1-22, wherein the enzyme has N125X3 substitution, or at a position corresponding thereto relative to SEQ ID NO: 1 .

24. The method of any one of claims 1-23, wherein Xi is selected from G, A, V, L, I, and P.

25. The method of any one of claims 1-24, wherein X2 is selected from K, R, and H.

26. The method of any one of claims 1-25, wherein X3 is selected K, R, and H.

27. The method of claim 1-26, wherein: Xi is P and X2 is R.

28. The method of any one of claims 1-27, wherein the enzyme is capable of inserting a donor DNA at a TA dinucleotide site.

29. The method of any one of claims 1-28, wherein the enzyme is capable of inserting a donor DNA at a TTAA tetranucleotide site.

30. The method of any one of claims 1-29, wherein the enzyme comprises: a targeting element and an enzyme that is capable of inserting the donor DNA (e.g., a mobile element) comprising a gene, optionally at a TA dinucleotide site or a TTAA tetranucleotide site in a genomic safe harbor site (GSHS).

31. The method of any one of claims 1 -30, wherein the targeting element comprises one or more of a gRNA, optionally associated with a Cas enzyme, which is optionally catalytically inactive or a transcription activator-like effector (TALE).

32. The method of any one of claims 1 -31 , wherein the targeting element comprises a transcription activatorlike effector (TALE) DNA binding domain (DBD) and zinc fingers (ZF) DBD.

33. The method of any one of claims 1-32, wherein the targeting element comprises a Cas9 enzyme guide RNA complex.

34. The method of claim 1 -33, wherein the Cas9 enzyme guide RNA complex comprises a nuclease-deficient dCas9 guide RNA complex.

35. The method of any one of claims 1-34, wherein the GSHS is in an open chromatin location in a chromosome.

36. The method of any one of claims 1-35, wherein the GSHS is selected from adeno-associated virus site 1 (AAVS1), chemokine (C-C motif) receptor 5 (CCR5) gene, HIV-1 coreceptor, and human Rosa26 locus.

37. The method of any one of claims 1-36, wherein the enzyme and the targeting element are connected.

38. The method of any one of claims 1-37, wherein the enzyme and the targeting element are fused to one another or linked via a linker to one another.

39. The method of claim 1 -38, wherein the linker is a flexible linker.

40. The method of claim 1-39, wherein the flexible linker is substantially comprised of glycine and serine residues, optionally wherein the flexible linker comprises (Gly4Ser)n, where n is from about 1 to about 12.

41. The method of claim 1-40, wherein the flexible linker is of about 20, or about 30, or about 40, or about 50, or about 60 amino acid residues.

42. The composition of any one of claims 1-41 , wherein the one or more nucleic acid-based agents comprise a donor DNA or transgene or mobile element.

43. The method of any one of claims 1 -42, wherein the donor DNA or transgene or mobile element comprises a gene encoding a complete polypeptide.

44. The method of any one of claims 1 -43, wherein the donor DNA or transgene or mobile element comprises a gene which is defective or substantially absent in a disease state.

45. The method of any one of claims 1 -44, wherein the donor DNA or transgene or mobile element is flanked by one or more ends.

46. The method of any one of claims 1-45, wherein the donor DNA or transgene or mobile element has a size of up to about 10 kb, or up to about 12 kb, or up to about 15 kb, or about 7.5 to about 15 kb, or about 10 to about 15 kb.

47. The composition of any one of claims 1-46, wherein the one or more nucleic acid-based agents is in the form of RNA.

48. The composition of claim 1-47, wherein the RNA is or comprises messenger RNA (mRNA).

49. The composition of claim 1-48, wherein the mRNA is or comprises modified mRNA (mmRNA).

50. The composition of claim 1-49, wherein the mmRNA comprises one or more of a 5’-m7G cap (capO, cap1 , or cap2), a pseudouridine or n-methyl-pseudouridine substitution, and a poly-A tail of about 30, or of about 50, or of about 100, or of about 150 nucleotides in length.

51. The composition of any one of claims 1-50, wherein the one or more nucleic acid-based agents is in the form of DNA.

52. The composition of claim 1-51 , wherein the DNA is or comprises plasmid DNA or miniplasmid DNA.

53. The composition of claim 1-52, wherein the plasmid DNA has a size of up to about 10 kb, or up to about 12 kb, or up to about 15 kb, or about 7.5 to about 15 kb, or about 10 to about 15 kb.

54. The composition of any one of claims 1-53, wherein the nucleic acid-based agent comprises both: a nucleic acid encoding a mobile element enzyme and a donor DNA or transgene or mobile element.

55. The composition of any one of claims 1-54, wherein the nucleic acid-based agent comprises both RNA and DNA.

56. The composition of any one of claims 1-55, wherein the nucleic acid-based agent comprises both mRNA and DNA.

57. The composition of claim 1-56, wherein the weight to weight ratio of DNA : mRNA is from about 10:1 to about 1 : 10, or about 10 : about 1 , or about 9 : about 1 , or about 8 : about 1 , or about 7 : about 1 , or about6 : about 1 , or about 5 : about 1 , or about 4 : about 1 , or about 3 : about 1 , or about 2 : about 1 , or about1 : about 1 , or about 1 : about 2, or about 1 : about 3, or about 1 : about 4, or about 1 : about 5, or about1 : about 6, or about 1 : about 7, or about 1 : about 8, or about 1 : about 9, or about 1 : about 10.

58. The composition of any one of claims 1-57, wherein the nucleic acid-based agent comprises both mmRNA and plasmid DNA.

59. The composition of any one of claims 1-58, wherein the nucleic acid-based agent comprises both: a nucleic acid encoding a mobile element enzyme in the form of mmRNA and a donor DNA or transgene or mobile element in the form of plasmid DNA.

60. The composition of any one of claims 1-59, wherein the nucleic acid-based agent comprises both: a nucleic acid encoding a mobile element enzyme in the form of mmRNA and a donor DNA or transgene or mobile element in the form of plasmid DNA and the weight to weight ratio of plasmid DNA: mmRNA is about 5 : about 1, or about 2 : about 1 , or about 1 : about 1 , or about 1 : about 2, or about 1 : about 5.

61. The composition of any one of claims 1-60, wherein the nucleic acid-based agent comprises both: a nucleic acid encoding a mobile element enzyme in the form of mmRNA and a donor DNA or transgene or mobile element in the form of plasmid DNA and the weight to weight ratio of plasmid DNA : mmRNA is about 2 : about 1 .

62. The composition of any one of claims 1 -61 , wherein the composition provides high encapsulation of the nucleic acid-based agents.

63. The composition of any one of claims 1 -62, wherein the composition provides an encapsulation efficiency of greater than about 90%, or greater than about 95%, or greater than about 98%.

64. The composition of any one of claims 1-63, wherein the composition provides low polydispersity.

65. The composition of any one of claims 1-64, wherein the composition is an LNP having a particle size of about 60 to about 200 nm, or about 100 to about 150 nm, or about 60 to about 120 nm.

66. The composition of any one of claims 1-65, wherein the composition is suitable for systemic delivery in a subject.

67. The composition of any one of claims 1-66, wherein the composition is suitable for intravenous, subcutaneous, or intraperitoneal delivery in a subject.

68. The composition of any one of claims 1-67, wherein the composition is suitable for local delivery in a subject.

69. The composition of any one of claims 1 -68, wherein the composition is suitable for subretinal delivery in a subject.

70. A method for introducing a nucleic acid into a cell, the method comprising contacting the cell with a composition of any one of claims 1-69.

71. A method for ocular delivery of nucleic acid, the method comprising ocularly administering to a subject a composition of any one of claims 1-69.

72. A method for the in vivo delivery of a nucleic acid, the method comprising administering to a subject a composition of any one of claims 1-69.

73. A method for treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition of any one of claims 1-69.

74. A method for treating an ocular disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition of any one of claims 1- 69.

75. A composition comprising:(a) one or more nucleic acid-based agents, and(b) one or more lipids, the one or more lipids comprising:(i) a delivery lipid comprising about 35 mol % of the total lipid present;(ii) cholesterol comprising about 50 mol % of the total lipid present;(iii) a helper lipid comprising about 10 mol % of the total lipid present; and(iv) a PEGylated lipid comprising about 5 mol % of the total lipid present.

76. The composition of claim 75, wherein the delivery lipid is SSOP.

77. The composition of claim 75, wherein the helper lipid is DOPC.

78. The composition of claim 75, wherein the PEGylated lipid is PEG-2k DMG.

79. The composition of claim 75, wherein: the delivery lipid is SSOP; the helper lipid is DOPC; and the PEGylated lipid is PEG-2k DMG.

80. The composition of claim 75, wherein:(a) one or more nucleic acid-based agents, and(b) one or more lipids, the one or more lipids comprising:(i) the delivery lipid is SSOP, comprising about 35.7 mol % of the total lipid present,(ii) cholesterol comprising about 49.8 mol % of the total lipid present,(iii) the helper lipid is DOPC, comprising about 9.6 mol % of the total lipid present, and(iv) the PEGylated lipid is PEG-2K DMG, comprising about 4.9 mol % of the total lipid present.

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